Berk2006-Sequences

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Sequence files in ApE format

download ApE


File:PAC997.str
File:PSB1A2.str
File:PSB1A3.str
File:PSB1AK3.str

garbage section 1

#F54D70 #5DFC0A #48D1CC #FF5333 #CC00FF #EEC900

garbage section 2

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<div class=Section1>

<p class=MsoNormal>072506</p>

<p class=MsoNormal>I’ve decided to keep just one notebook from here on.<span style='mso-spacerun:yes'> </span>It gets too confusing to figure out what experiments/constructions are in which file.<span style='mso-spacerun:yes'> </span>It would be easier just to have it all in one place and be searchable.<span style='mso-spacerun:yes'> </span>I think what I’ll do is keep separate word files with all the embedded bits and pieces and then put just text on the <span class=SpellE>wiki</span> page.<span style='mso-spacerun:yes'> </span>I’m repeating the last few days here for convenience sake.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><st1:date Year="2006" Day="8" Month="7">Saturday, July 08,

2006</st1:date></p>

<p class=MsoNormal>I <span class=SpellE>mini’d</span> and worked up for sequencing Bca9004, Bca9009, Bca9018, and Bca9021.<span style='mso-spacerun:yes'> </span>At one point I had a <span class=SpellE>mixup</span> of 9009 calling it 9003.<span style='mso-spacerun:yes'> </span>For some reason I had labeled the plate 9003.<span style='mso-spacerun:yes'> </span>I think I fixed it all—the -80 <span class=GramE>stock</span> and mini are labeled 9009.<span style='mso-spacerun:yes'> </span><span class=SpellE>Anywho</span>, we’ll send that out Monday.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>I did a bunch of <span class=SpellE>subcloning</span> today.<span style='mso-spacerun:yes'> </span>The <span class=SpellE>oligos</span> to do <span class=SpellE>TriR</span> and <span class=SpellE>CmR</span> cassettes came, so the 4 parts involving those were in the mix.<span style='mso-spacerun:yes'> </span>Total I did 11 <span class=SpellE>subclones</span>:</p>

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<p class=MsoNormal>There was some gel purification and analytical <span class=SpellE>PCRs</span>.<span style='mso-spacerun:yes'> </span>They were assigned letter codes according to:</p>

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<p class=MsoNormal>The gel goes: 1015 <span class=SpellE>pcr</span>; 1016 <span class=SpellE>pcr</span>; b0015 <span class=SpellE>pcr</span>; mw; b0015 <span class=SpellE>pcr</span>; A; f; F; e; ; d; d; d; d:</p>

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<p class=MsoNormal>Sizes all look reasonable, but I didn’t check all that carefully.<span style='mso-spacerun:yes'> </span>The band pattern of the “d” digest of Bca9008 (<span class=SpellE>BglII/MfeI</span>) confirms the presence of the restriction sites.<span style='mso-spacerun:yes'> </span>I don’t plan to sequence 9008 <span class=GramE>directly,</span> I’ll get a de factor read from parts put in.<span style='mso-spacerun:yes'> </span>I put in <span class=SpellE>wbbL</span>, <span class=SpellE>invasin</span>, and 2 <span class=SpellE>rfbC</span> hits today.<span style='mso-spacerun:yes'> </span>So, these cassettes are RBS-ORF composites, no terminators.<span style='mso-spacerun:yes'> </span>Regardless of the sequence of J01008, I’m going with this clone.<span style='mso-spacerun:yes'> </span>If there are points, I’ll change the model sequence.<span style='mso-spacerun:yes'> </span>The last 2 parts are part-building parts.<span style='mso-spacerun:yes'> </span>9028 is like 1008, but it includes a <span class=SpellE>dblTerm</span> downstream of the <span class=SpellE>BglII/MfeI</span> insertion sites.<span style='mso-spacerun:yes'> </span>So, the product of conversion is an RBS-ORF-TT composite.<span style='mso-spacerun:yes'> </span>Part 9027 is for conversion of <span class=SpellE>biobricks</span> into pBAC874t via a <span class=SpellE>NotI/EcoRI</span> <span class=SpellE>subclone</span>.<span style='mso-spacerun:yes'> </span>It destroys the <span class=SpellE>NotI</span> site between <span class=SpellE>EcoRI</span> and <span class=SpellE>XbaI</span> so that the one between <span class=SpellE>SpeI</span> and <span class=SpellE>PstI</span> is unique.<span style='mso-spacerun:yes'> </span>It also puts in <span class=SpellE>dblTerm</span>.<span style='mso-spacerun:yes'> </span>To put parts into pBca9027, I would <span class=SpellE>subclone</span> into <span class=SpellE>SpeI/AlwNI</span>, <span class=GramE>then</span> transfer <span class=SpellE>NotI/EcoRI</span> into the BAC.<span style='mso-spacerun:yes'> </span>I’ll be using that guy for the O-antigen AND gate.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><st1:date Year="2006" Day="12" Month="7">Wednesday, July 12,

2006</st1:date></p>

<p class=MsoNormal><span class=GramE>More <span class=SpellE>subcloning</span> fun.</span><span style='mso-spacerun:yes'> </span>The 4 <span class=SpellE>subclones</span> into pBca1008 didn’t go so well:</p>

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<p class=MsoNormal>They all map funky, so I’m redoing all that stuff with proper cut and paste with gel purification.<span style='mso-spacerun:yes'> </span>I got good clones of all the constructs from the other day except Bca1015.<span style='mso-spacerun:yes'> </span>I’m just redoing that guy.<span style='mso-spacerun:yes'> </span>All the <span class=SpellE>CmR</span>-related <span class=SpellE>subclones</span> look fabulous.</p>

<p class=MsoNormal>I’m not going to sequence the <span class=SpellE>CmR</span> and <span class=SpellE>TriR</span> basic parts at <span class=GramE>all,</span> I’m going for de facto sequencing at a later point.<span style='mso-spacerun:yes'> </span>I want to proceed all the way to the [FRT<span class=GramE>][</span>*R][FRT] parts <span class=SpellE>asap</span> to test FRT function. I also got in the <span class=SpellE>PspOMI</span> enzyme, so I could do the Bca1007 and Bca1009 digests.<span style='mso-spacerun:yes'> </span>Both digested fine, so they are ok.<span style='mso-spacerun:yes'> </span>So, I proceeded with the following <span class=SpellE>subclones</span>:</p>

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<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>….and set up the digests as:</p>

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<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>The gel below goes <span class=SpellE>AaBbCcDG</span>, all are good.<span style='mso-spacerun:yes'> </span>This gel confirms the general size of the <span class=SpellE>CmR</span>-FRT, <span class=SpellE>CmR</span>, and <span class=SpellE>TriR</span>-FRT parts.</p>

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<p class=MsoNormal>The gels on <span class=SpellE>d<span class=GramE>,g,h,i,j</span></span> etc. initially didn’t go so well.<span style='mso-spacerun:yes'> </span>I was able to get only <span class=SpellE>d<span class=GramE>,i</span></span>, and j out of that gel, the rest looked like shit.<span style='mso-spacerun:yes'> </span>I didn’t even save the gel.<span style='mso-spacerun:yes'> </span>Originally, I was doing a different set of <span class=SpellE>subclones</span>, so that’s not here, it included <span class=SpellE>mgrB</span>.<span style='mso-spacerun:yes'> </span>For the remaining ones on the list above, I went with inserts either from PCR (<span class=SpellE>flu<span class=GramE>,fdhf</span></span>, wbb992) or existing digests (<span class=SpellE>AraInv</span>).<span style='mso-spacerun:yes'> </span>So the first list above is the “correct” list.<span style='mso-spacerun:yes'> </span>I’ll need to make new maps of some of this—I don’t have a map for flu or <span class=SpellE>fdhF</span>, and wbb992 will replace the <span class=SpellE>TetWbb</span>-derived <span class=SpellE>subclone</span>.<span style='mso-spacerun:yes'> </span>I just relegated the 1015 basic <span class=SpellE>TriR</span> part, and plated on both Amp and Tri/Amp to make sure I got <span class=SpellE>cfus</span> in case there is something weird (like <span class=SpellE>TetR</span> colony reduction) with <span class=SpellE>Trimethoprim</span> resistance.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>I’ve sent a bunch of clones from the last batch for sequencing.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><st1:date Year="2006" Day="12" Month="7">Wednesday, July 12,

2006</st1:date></p>

<p class=MsoNormal>I got sequencing back from the stuff I sent, most look good:</p>

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<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>There was an error in the model for Bca9028.<span style='mso-spacerun:yes'> </span>I had part b0015 in pSB1A2, and in reality it is in pSB1AK3 (and the registry says so).<span style='mso-spacerun:yes'> </span>When I remade the model file with the correct starting sequence, the sequencing matched the new model for what I’m going to call pSB1A0-Bca9028.<span style='mso-spacerun:yes'> </span><span class=GramE>All good, no biggie.</span><span style='mso-spacerun:yes'> </span>It is not a pSB1AK3 as the <span class=SpellE>KanR</span> marker would not be transferred by the sub method (I don’t think…maybe check it if <span class=GramE>it<span style='mso-spacerun:yes'> </span>becomes</span> an issue).<span style='mso-spacerun:yes'> </span>Part pSB1A2-Bca9009 matches the model, but the read didn’t make it past the part junction.<span style='mso-spacerun:yes'> </span>I’ll need to do confirmation by PCR rather than sequencing.<span style='mso-spacerun:yes'> </span><span class=GramE>pBca9027</span> is not correct, I need to examine more clones.<span style='mso-spacerun:yes'> </span>Unfortunately, this list doesn’t get me to more <span class=SpellE>subclones</span>, so tomorrow will just be characterization of all sorts of stuff.<span style='mso-spacerun:yes'> </span>Mainly, I need to confirm the FRT-*-FRT constructs and make sure that pCP20 excises the fragment properly.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><st1:date Year="2006" Day="13" Month="7">Thursday, July 13,

2006</st1:date></p>

<p class=MsoNormal>I threw out the sequenced clone of Bca9027 and grew up 4 more.<span style='mso-spacerun:yes'> </span>I did characterization of various things:</p>

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<p class=MsoNormal>The gels (<span class=SpellE>PCRs</span> 1,2,3,Mw,a,b,c,d,e,f,g,h) are:</p>

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<p class=MsoNormal>The Bca9009 is confirmed (combination of sequencing and PCR “1”).<span style='mso-spacerun:yes'> </span>The first half of 9010 is confirmed, I think I’ll do some mapping for the <span class=SpellE>Tnp</span> half.<span style='mso-spacerun:yes'> </span>The PCR failed, but it is probably right—just too long for a 2K <span class=SpellE>pcr</span>.<span style='mso-spacerun:yes'> </span>At least I hope so….we’ll find out tomorrow.<span style='mso-spacerun:yes'> </span>The results of mapping go:</p>

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 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Bca9007<o:p></o:p></span></p>
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 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>[FRT][<span
 class=SpellE>CmR</span>][FRT]<o:p></o:p></span></p>
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 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>consistent<o:p></o:p></span></p>
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 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Bca9015<o:p></o:p></span></p>
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 <td width=132 nowrap valign=bottom style='width:98.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>[<span
 class=SpellE>Psal</span>]<o:p></o:p></span></p>
 </td>
 <td width=224 nowrap valign=bottom style='width:167.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>consistent<o:p></o:p></span></p>
 </td>
</tr>
<tr style='mso-yfti-irow:2;height:12.75pt'>
 <td width=64 nowrap valign=bottom style='width:47.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Bca9026<o:p></o:p></span></p>
 </td>
 <td width=132 nowrap valign=bottom style='width:98.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>[Flu]<o:p></o:p></span></p>
 </td>
 <td width=224 nowrap valign=bottom style='width:167.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>consistent<o:p></o:p></span></p>
 </td>
</tr>
<tr style='mso-yfti-irow:3;height:12.75pt'>
 <td width=64 nowrap valign=bottom style='width:47.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Bca9014<o:p></o:p></span></p>
 </td>
 <td width=132 nowrap valign=bottom style='width:98.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>[wbbL1]<o:p></o:p></span></p>
 </td>
 <td width=224 nowrap valign=bottom style='width:167.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>consistent<o:p></o:p></span></p>
 </td>
</tr>
<tr style='mso-yfti-irow:4;height:12.75pt'>
 <td width=64 nowrap valign=bottom style='width:47.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Bca9013<o:p></o:p></span></p>
 </td>
 <td width=132 nowrap valign=bottom style='width:98.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>[inv1]<o:p></o:p></span></p>
 </td>
 <td width=224 nowrap valign=bottom style='width:167.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>off<o:p></o:p></span></p>
 </td>
</tr>
<tr style='mso-yfti-irow:5;height:12.75pt'>
 <td width=64 nowrap valign=bottom style='width:47.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Bca9031<o:p></o:p></span></p>
 </td>
 <td width=132 nowrap valign=bottom style='width:98.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>[P_sal-rfbC1]<o:p></o:p></span></p>
 </td>
 <td width=224 nowrap valign=bottom style='width:167.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>consistent<o:p></o:p></span></p>
 </td>
</tr>
<tr style='mso-yfti-irow:6;height:12.75pt'>
 <td width=64 nowrap valign=bottom style='width:47.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Bca9032<o:p></o:p></span></p>
 </td>
 <td width=132 nowrap valign=bottom style='width:98.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>[P_sal-rfbC2]<o:p></o:p></span></p>
 </td>
 <td width=224 nowrap valign=bottom style='width:167.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>consistent<o:p></o:p></span></p>
 </td>
</tr>
<tr style='mso-yfti-irow:7;mso-yfti-lastrow:yes;height:12.75pt'>
 <td width=64 nowrap valign=bottom style='width:47.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Bca9033<o:p></o:p></span></p>
 </td>
 <td width=132 nowrap valign=bottom style='width:98.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>[<span
 class=SpellE>fdhF</span>]<o:p></o:p></span></p>
 </td>
 <td width=224 nowrap valign=bottom style='width:167.8pt;padding:0in 5.4pt 0in 5.4pt;
 height:12.75pt'>
 <p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>consistent<o:p></o:p></span></p>
 </td>
</tr>

</table>

<p class=MsoNormal>So, all but <span class=SpellE>invasin</span> are ok.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><st1:date Year="2006" Day="15" Month="7">Saturday, July 15,

2006</st1:date></p>

<p class=MsoNormal>I got some sequencing results back:</p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1036" type="#_x0000_t75"

style='width:6in;height:57pt'>
<v:imagedata src="Installment1-start072506_files/image023.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=576 height=76 src="Installment1-start072506_files/image024.gif" v:shapes="_x0000_i1036"><![endif]></p>

<p class=MsoNormal>All are fine, didn’t get the full <span class=SpellE>rbs</span> for the <span class=SpellE>wbbL</span> <span class=SpellE>rbs</span>, but got some of it looking at the trace, and <span class=GramE>it’s</span> fine.<span style='mso-spacerun:yes'> </span><span class=GramE>Going with it.</span> </p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>I’ve done some <span class=SpellE>EcoRI/PstI</span> mapping of more clones of the ones that were problematic:</p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1037" type="#_x0000_t75"

style='width:171pt;height:160.5pt' o:allowoverlap="f">
<v:imagedata src="Installment1-start072506_files/image025.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=228 height=214 src="Installment1-start072506_files/image026.gif" v:shapes="_x0000_i1037"><![endif]></p>

<p class=MsoNormal>Bca9005-still wrong</p>

<p class=MsoNormal>Bca1015-still wrong</p>

<p class=MsoNormal>Bca9027-1<span class=GramE>,3,4</span>-good</p>

<p class=MsoNormal>Bca9027-2, wrong</p>

<p class=MsoNormal>Bca9013-1<span class=GramE>,2</span>-good</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>I’ll stock and sequence 9027-1 and 9013-1.<span style='mso-spacerun:yes'> </span>Holding onto the minis of the other 9027’s till get sequence confirmation.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><st1:date Year="2006" Day="19" Month="7">Wednesday, July 19,

2006</st1:date></p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1038" type="#_x0000_t75"

style='width:6in;height:16.5pt'>
<v:imagedata src="Installment1-start072506_files/image027.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=576 height=22 src="Installment1-start072506_files/image028.gif" v:shapes="_x0000_i1038"><![endif]></p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>The <span class=GramE>new#1 clones</span> of pBca9027 and pSB1A2-Bca9013 are fine.<span style='mso-spacerun:yes'> </span>The region downstream of <span class=SpellE>PstI</span> in 9027 still isn’t matching template, but it won’t affect the usability of the plasmid, so no worries.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>I did some mapping of recent <span class=SpellE>subclones</span>:</p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1039" type="#_x0000_t75"

style='width:431.25pt;height:24pt'>
<v:imagedata src="Installment1-start072506_files/image029.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=575 height=32 src="Installment1-start072506_files/image030.gif" v:shapes="_x0000_i1039"><![endif]></p>

<p class=MsoNormal>They go:</p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.5in;mso-list:l0 level1 lfo1; tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>1<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>pSB2K3-Bca9007-1</p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.5in;mso-list:l0 level1 lfo1; tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>2<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>pSB2K3-Bca9007-2</p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.5in;mso-list:l0 level1 lfo1; tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>3<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>pSB2K3-Bca9007-3</p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.5in;mso-list:l0 level1 lfo1; tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>4<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>pJ23006-Bca9034</p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.5in;mso-list:l0 level1 lfo1; tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>5<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>pSB1A2-Bca9005-1</p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.5in;mso-list:l0 level1 lfo1; tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>6<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>pSB1A2-Bca9005-2</p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.5in;mso-list:l0 level1 lfo1; tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>7<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>pSB1A2-Bca9010-1</p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.5in;mso-list:l0 level1 lfo1; tab-stops:list .75in'><![if !supportLists]><span style='mso-list:Ignore'>8<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span><![endif]>pSB1A2-Bca9010-2</p>

<p class=MsoNormal style='margin-left:.25in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='margin-left:.25in'>The mapping below is all <span class=SpellE>EcoRI/PstI</span>.<span style='mso-spacerun:yes'> </span>Clones 1 and 3 of pSB2K3-Bca9007 were bigger, healthier clones.<span style='mso-spacerun:yes'> </span>They appear to be the <span class=SpellE>cotransformed</span> guys, though.<span style='mso-spacerun:yes'> </span>Clone 2 was sickly but maps right.<span style='mso-spacerun:yes'> </span>The rest of these guys all map correct.<span style='mso-spacerun:yes'> </span>I did 1015 (<span class=SpellE>TriR</span> alone) in this batch.<span style='mso-spacerun:yes'> </span>It again didn’t give any <span class=SpellE>triR</span> colonies.<span style='mso-spacerun:yes'> </span><span class=GramE>Whatever.</span><span style='mso-spacerun:yes'> </span>I’m done with that for now I think.<span style='mso-spacerun:yes'> </span>Samantha made a <span class=SpellE>SpecR</span> basic part which is probably more user friendly.<span style='mso-spacerun:yes'> </span>Bca9010 went very smoothly this time, many <span class=SpellE>CmR/AmpR</span> colonies, and they mapped right.<span style='mso-spacerun:yes'> </span><span class=GramE>Will need to PCR map those, though to confirm the <span class=SpellE>Tnp</span> presence.</span></p>

<p class=MsoNormal style='margin-left:.25in'><!--[if gte vml 1]><v:shape id="_x0000_i1040"

type="#_x0000_t75" style='width:207pt;height:141.75pt'>
<v:imagedata src="Installment1-start072506_files/image031.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=276 height=189 src="Installment1-start072506_files/image032.jpg" v:shapes="_x0000_i1040"><![endif]></p>

<p class=MsoNormal style='margin-left:.25in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><st1:date Year="2006" Day="21" Month="7">Friday, July 21,

2006</st1:date></p>

<p class=MsoNormal>I got sequencing results for pSB1A2-Bca9005 (F-Tri-F).<span style='mso-spacerun:yes'> </span>It was perfect.<span style='mso-spacerun:yes'> </span>I have still been unable to make a <span class=SpellE>TriR</span> basic part.<span style='mso-spacerun:yes'> </span><span class=GramE>Very weird there, since the composite works great.</span><span style='mso-spacerun:yes'> </span>BTW, this is also <span class=SpellE>defacto</span> sequencing of Bca9002 since the read got the entire cassette.<span style='mso-spacerun:yes'> </span>All good<span class=GramE>,<span style='mso-spacerun:yes'> </span>both</span> <span class=SpellE>FRT’s</span> present.<span style='mso-spacerun:yes'> </span>I also got sequence on pJ23006-Bca9034, and it too is perfect.</p>

<p class=MsoNormal style='margin-left:.5in;tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><span lang=PT-BR style='font-size:8.0pt;font-family:"Courier New";mso-ansi-language: PT-BR'>jca293 <span style='mso-tab-count:1'> </span>072006 <span style='mso-tab-count:1'> </span>pSB1A2-Bca9005 <span style='mso-tab-count: 1'> </span>1 <span style='mso-tab-count:1'> </span>ca998 <span style='mso-tab-count:1'> </span>perfect<o:p></o:p></span></p>

<p class=MsoNormal style='margin-left:.5in;tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><span lang=PT-BR style='font-size:8.0pt;font-family:"Courier New";mso-ansi-language: PT-BR'>jca294 <span style='mso-tab-count:1'> </span>072006 <span style='mso-tab-count:1'> </span>pJ23006-Bca9034 <span style='mso-tab-count: 1'> </span>1 <span style='mso-tab-count:1'> </span>ca998 <span style='mso-tab-count:1'> </span>perfect</span><span lang=PT-BR style='font-family:"Courier New";mso-ansi-language:PT-BR'><o:p></o:p></span></p>

<p class=MsoNormal><span lang=PT-BR style='mso-ansi-language:PT-BR'><o:p>&nbsp;</o:p></span></p>

<p class=MsoNormal><span lang=PT-BR style='mso-ansi-language:PT-BR'><o:p>&nbsp;</o:p></span></p>

<p class=MsoNormal>To confirm pSB1A2-Bca9010 (<span class=SpellE>CmR.Tnp</span>), I did PCR w/ 998 and 9014R, expected size 2721 <span class=SpellE>bp</span>.<span style='mso-spacerun:yes'> </span>The third lane on the gel below is that.<span style='mso-spacerun:yes'> </span><span class=GramE>Looks solid.</span></p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1041" type="#_x0000_t75"

style='width:92.25pt;height:120pt'>
<v:imagedata src="Installment1-start072506_files/image033.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=123 height=160 src="Installment1-start072506_files/image034.jpg" v:shapes="_x0000_i1041"><![endif]><o:p></o:p></p>

<p class=MsoNormal>I wanted to confirm the activity of FRT (if I could) at this stage, so I moved the Bca9007 cassette into pSB2K3 and then transformed pCP20 comp with that guy.<span style='mso-spacerun:yes'> </span><span class=GramE>If the thing <span class=SpellE>flps</span>, the <span class=SpellE>EcoRI/PstI</span> region changes size.</span><span style='mso-spacerun:yes'> </span>The expected fragments would be:<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.5in;tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><span style='font-size:8.0pt;font-family:"Courier New"'>No <span class=SpellE>flp</span><span style='mso-tab-count:1'> </span>pSB2K3-Bca9007<span style='mso-tab-count: 1'> </span>3446+1957<o:p></o:p></span></p>

<p class=MsoNormal style='margin-left:.5in;tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><span style='font-size:8.0pt;font-family:"Courier New"'>FRT deletion<span style='mso-tab-count:2'> </span>3446+1013<o:p></o:p></span></p>

<p class=MsoNormal>The bands in the middle are <span class=GramE>a <span class=SpellE>miniprep</span></span> of a clone grown ON in just <st1:State><st1:place><span

 class=SpellE>kan</span></st1:place></st1:State> (to clear pCP20, maintain the

pSB2K3).<span style='mso-spacerun:yes'> </span>The result is a little fishy.<span style='mso-spacerun:yes'> </span>The digest btw is <span class=SpellE>XhoI/PstI</span> in neb2.<span style='mso-spacerun:yes'> </span>It looks like the 3446 band is there, and the plasmid is a partial digest.<span style='mso-spacerun:yes'> </span>The fragment band that would correspond to the F-Cm-F fragment is too big.<span style='mso-spacerun:yes'> </span>It’s either a map error, or some non-expected recombination occurring.<span style='mso-spacerun:yes'> </span>I’m not convinced this is a valid experiment—this might not fly to do it this way.<span style='mso-spacerun:yes'> </span>The small band around 600 <span class=SpellE>bp</span> isn’t an expected fragment either.<span style='mso-spacerun:yes'> </span>Not sure how that comes to be.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>The first band on the gel comes from transforming pKD908b/pir116 with Bca9010.<span style='mso-spacerun:yes'> </span>Those cells did not look so healthy on the plate, <span class=SpellE>kindov</span> small.<span style='mso-spacerun:yes'> </span>I grew one up in just Amp, and it looks like the cells just kicked out the R6K plasmid.<span style='mso-spacerun:yes'> </span>BTW, that’s a <span class=SpellE>SpeI</span> digest which would <span class=SpellE>linearize</span> all <span class=SpellE>relavent</span> plasmids.<span style='mso-spacerun:yes'> </span>So, that’s no confirmation of <span class=SpellE>Tnp</span> function.<span style='mso-spacerun:yes'> </span>I would really like to see some evidence of FRT and <span class=SpellE>Tnp</span> function before proceeding, but I’m not sure how else to assay it.<span style='mso-spacerun:yes'> </span>I’m only 2 steps away from being able to test the <span class=SpellE>Tnp</span> final construct.<span style='mso-spacerun:yes'> </span>So, I’ll just test at the end and hope for the best.<span style='mso-spacerun:yes'> </span>I think I’ll also just have to wing the FRT stuff until there is a genome-integrated cassette.</p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'>I <span class=SpellE>miniprepped</span> and -80’d clones of the following constructs:</p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><!--[if gte vml 1]><v:shape

id="_x0000_i1042" type="#_x0000_t75" style='width:431.25pt;height:18.75pt'>
<v:imagedata src="Installment1-start072506_files/image035.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=575 height=25 src="Installment1-start072506_files/image036.gif" v:shapes="_x0000_i1042"><![endif]></p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'>I’ll do de facto characterization of those since they each manifest a phenotype.</p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'>I did a series of <span class=SpellE>subclones</span> today…</p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><!--[if gte vml 1]><v:shape

id="_x0000_i1043" type="#_x0000_t75" style='width:6in;height:75pt'>
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</v:shape><![endif]--><![if !vml]><img width=576 height=100 src="Installment1-start072506_files/image038.gif" v:shapes="_x0000_i1043"><![endif]></p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><span class=SpellE>Hh</span> and Ff insert S03155 looked like crap, so I grew up more to <span class=SpellE>miniprep</span> and <span class=SpellE>redigest</span>.<span style='mso-spacerun:yes'> </span>The large fragments for each were fine.</p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><st1:date Year="2006" Day="24" Month="7">Monday, July 24, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:1.25in 153.0pt 3.5in 297.0pt 5.0in'><!--[if gte vml 1]><v:shape

id="_x0000_i1044" type="#_x0000_t75" style='width:270pt;height:183.75pt'
o:allowoverlap="f">
<v:imagedata src="Installment1-start072506_files/image039.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=360 height=245 src="Installment1-start072506_files/image040.gif" v:shapes="_x0000_i1044"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span lang=PT-BR style='font-size:8.0pt;font-family:"Courier New";color:red; mso-ansi-language:PT-BR'>pBca1020-bca9015<span style='mso-tab-count:1'> </span>perfect<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span lang=PT-BR style='font-size:8.0pt;font-family:"Courier New";color:red; mso-ansi-language:PT-BR'>pBca1020-Bca9022-1<span style='mso-tab-count:1'> </span>perfect<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span class=GramE><span style='font-size:8.0pt;font-family:"Courier New"'>pBca1020-Bca9022-2</span></span><span style='font-size:8.0pt;font-family:"Courier New"'><span style='mso-tab-count: 1'> </span>wrong<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span class=GramE><span style='font-size:8.0pt;font-family:"Courier New"'>pBca1020-Bca9022-3</span></span><span style='font-size:8.0pt;font-family:"Courier New"'><span style='mso-tab-count: 1'> </span>wrong<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span class=GramE><span style='font-size:8.0pt;font-family:"Courier New"'>pSB1A2-Bca9006</span></span><span style='font-size:8.0pt;font-family:"Courier New"'><span style='mso-tab-count: 1'> </span>wrong<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span class=GramE><span style='font-size:8.0pt;font-family:"Courier New";color:red'>pSB1A2-Bca9008</span></span><span style='font-size:8.0pt;font-family:"Courier New";color:red'><span style='mso-tab-count:1'> </span>perfect<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span lang=PT-BR style='font-size:8.0pt;font-family:"Courier New";color:red; mso-ansi-language:PT-BR'>pBca1020-Bca9036-1<span style='mso-tab-count:1'> </span>perfect<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span lang=PT-BR style='font-size:8.0pt;font-family:"Courier New";mso-ansi-language: PT-BR'>pBca1020-Bca9036-2<span style='mso-tab-count:1'> </span>perfect<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span class=GramE><span style='font-size:8.0pt;font-family:"Courier New";color:red'>pSB1AK3-Bca9037</span></span><span style='font-size:8.0pt;font-family:"Courier New";color:red'><span style='mso-tab-count:1'> </span>perfect<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span class=GramE><span style='font-size:8.0pt;font-family:"Courier New"'>pSB1AK3-Bca9038</span></span><span style='font-size:8.0pt;font-family:"Courier New"'><span style='mso-tab-count: 1'> </span>extra band (<span class=SpellE>contam</span>?)<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt 3.5in 297.0pt 5.0in'><span style='font-size:8.0pt;font-family:"Courier New"'><o:p>&nbsp;</o:p></span></p>

<p class=MsoNormal style='margin-left:.25in'><span class=GramE>Keeping and -80’ing the ones in red.</span><span style='mso-spacerun:yes'> </span><span class=GramE>Growing up more 9006 and 9038.</span></p>

<p class=MsoNormal style='margin-left:.25in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='margin-left:.25in'><st1:date Year="2006" Day="25" Month="7">Tuesday, July 25, 2006</st1:date></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'>I <span class=SpellE>mini’d</span> and mapped some more constructs.<span style='mso-spacerun:yes'> </span>I don’t know what’s up with the <span class=SpellE>trimethoprim</span> marker, but I’m starting to think I should just ditch it.<span style='mso-spacerun:yes'> </span>Yeah, I think I’m going to do that and maybe replace its use with the <span class=SpellE>specR</span> parts.<span style='mso-spacerun:yes'> </span>This just isn’t happening.</p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><!--[if gte vml 1]><v:shape

id="_x0000_i1045" type="#_x0000_t75" style='width:219.75pt;height:219.75pt'
o:allowoverlap="f">
<v:imagedata src="Installment1-start072506_files/image041.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=293 height=293 src="Installment1-start072506_files/image042.gif" v:shapes="_x0000_i1045"><![endif]></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><span class=GramE>pSB1A2-Bca9001</span><span style='mso-tab-count:1'> </span>perfect<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><span class=GramE>pSB1A2-Bca9006-1</span><span style='mso-tab-count:1'> </span>weird<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><span class=GramE>pSB1A2-Bca9006-2</span><span style='mso-tab-count:1'> </span>weird<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><span class=GramE>pSB1A2-Bca9006-3</span><span style='mso-tab-count:1'> </span>weird<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><span class=GramE>pSB1A2-Bca9006-4</span><span style='mso-tab-count:1'> </span>weird<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><span class=GramE>pSB1A2-Bca9029</span><span style='mso-tab-count:1'> </span>weird<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><span class=GramE>pSB1AK3-Bca9038-1</span><span style='mso-tab-count:1'> </span>right, I think<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.25in;tab-stops:2.25in'><span class=GramE>pSB1AK3-Bca9038-2</span><span style='mso-tab-count:1'> </span>wrong, I think<o:p></o:p></p>

<p class=MsoNormal style='margin-left:.25in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='margin-left:.25in'>The pBca1020 series <span class=GramE>plasmids is</span> working out just fine now.<span style='mso-spacerun:yes'> </span>Initially I tried putting the RFP into the <span class=SpellE>Salicylate</span> promoter part (9015).<span style='mso-spacerun:yes'> </span>I got no red colonies, so I put it into R0040 which gave the expected phenotype.<span style='mso-spacerun:yes'> </span>I took one of those guys and <span class=SpellE>subcloned</span> in the <span class=GramE>IPTG(</span>9022) part and the Sal(9015).<span style='mso-spacerun:yes'> </span>The <span class=SpellE>sal</span> ones came out white w/o <span class=SpellE><span class=GramE>sal</span></span><span class=GramE>,</span> the IPTG ones were red without <span class=SpellE>sal</span>.<span style='mso-spacerun:yes'> </span>I grew up the Sal clone with 100 <span class=SpellE>ug/mL</span> <span class=SpellE>sal</span>, and the cell pellet was pinky, but way lower expression than with <span class=SpellE><span class=GramE>tet</span></span> promoter.<span style='mso-spacerun:yes'> </span><span class=GramE>A little weird.</span><span style='mso-spacerun:yes'> </span>I was worried that the IPTG part wasn’t inducing, so I tested the 1020 with an induction series—it’s just very leaky at high copy, but that should settle down at single copy I think (below).<span style='mso-spacerun:yes'> </span>The half-max seems to be around 5 <span class=SpellE>ug/mL</span> of IPTG.<span style='mso-spacerun:yes'> </span>Good enough, going to proceed with putting on <span class=SpellE>wbbL</span>.</p>

<p class=MsoNormal style='margin-left:.25in'><!--[if gte vml 1]><v:shape id="_x0000_i1046"

type="#_x0000_t75" style='width:404.25pt;height:141pt'>
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<p class=MsoNormal style='margin-left:.25in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='margin-left:.25in'>I <span class=SpellE>subcloned</span> the extension product for the 1022 library into the <span class=SpellE>sal</span> construct (pBca1020-Bca9015) and got ~5% pinkish colonies.<span style='mso-spacerun:yes'> </span>Matt grew up 96 pinks, 96 whites, and I <span class=SpellE>reastreaked</span> 16 too-dense clones that were very red.<span style='mso-spacerun:yes'> </span>We’ll sort through that with the <span class=SpellE>tecan</span> tomorrow.</p>

<p class=MsoNormal style='margin-left:.25in'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='margin-left:.25in'>I did several new <span class=SpellE>subclones</span> today:</p>

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type="#_x0000_t75" style='width:6in;height:34.5pt'>
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<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><st1:date Month="7" Day="26" Year="2006">Wednesday, July 26,

2006</st1:date></p>

<p class=MsoNormal>I did PCR to confirm the composition of Bca9001.<span style='mso-spacerun:yes'> </span>They are:</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>KB001/ca641F<span style='mso-tab-count:1'> </span>TT<span style='mso-tab-count:1'> </span>2048bp</p>

<p class=MsoNormal>KB009/ca641F<span style='mso-tab-count:1'> </span><span class=SpellE>Ptet</span><span style='mso-tab-count:1'> </span>1884bp</p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1048" type="#_x0000_t75"

style='width:87pt;height:133.5pt'>
<v:imagedata src="Installment1-start072506_files/image047.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=116 height=178 src="Installment1-start072506_files/image048.jpg" v:shapes="_x0000_i1048"><![endif]></p>

<p class=MsoNormal>Looks solid, proceeding with the <span class=SpellE>subclones</span> to do <span class=SpellE>neuD</span> and <span class=SpellE>wbbL</span> <span class=SpellE>knockins</span>:</p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1049" type="#_x0000_t75"

style='width:6in;height:17.25pt'>
<v:imagedata src="Installment1-start072506_files/image049.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=576 height=23 src="Installment1-start072506_files/image050.gif" v:shapes="_x0000_i1049"><![endif]></p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal>Yesterdays <span class=SpellE>subclones</span> all gave tons of colonies, growing up one of each.</p>

<p class=MsoNormal>The 1022 library gave the following profile.<span style='mso-spacerun:yes'> </span>This is FluorRFP/OD600.<span style='mso-spacerun:yes'> </span>The whiter ones all were from white colonies, redder from red, so that’s <span class=SpellE>consistant</span>.<span style='mso-spacerun:yes'> </span>The parent construct (<span class=SpellE>Psal</span>) comes out around 55 on this chart.<span style='mso-spacerun:yes'> </span><span class=SpellE>Ptet</span> is at the far right, comparable in activity to the best 3 hits.<span style='mso-spacerun:yes'> </span>I <span class=SpellE>restreaked</span> multiple colonies as well, grew up some of those to assay tomorrow.<span style='mso-spacerun:yes'> </span>Matt grew about 18 clones across the spectrum of activity to assay tomorrow by <span class=SpellE>Tecan/Cytometry</span> and then mini, stock, seq.</p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1050" type="#_x0000_t75"

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</v:shape><![endif]--><![if !vml]><img width=525 height=249 src="Installment1-start072506_files/image052.gif" v:shapes="_x0000_i1050"><![endif]></p>

<p class=MsoNormal><st1:date Year="2006" Day="27" Month="7">Thursday, July 27,

2006</st1:date></p>

<p class=MsoNormal><o:p>&nbsp;</o:p></p>

<p class=MsoNormal><!--[if gte vml 1]><v:shape id="_x0000_i1051" type="#_x0000_t75"

style='width:132pt;height:195pt' o:allowoverlap="f">
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</v:shape><![endif]--><![if !vml]><img width=176 height=260 src="Installment1-start072506_files/image054.gif" v:shapes="_x0000_i1051"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pSB1A2-Bca9023</span><span style='mso-tab-count:1'> </span>wrong<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pBca9020-Bca9030</span><span style='mso-tab-count:1'> </span>good<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pSB1A2-Bca9020</span><span style='mso-tab-count:1'> </span>good<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pSB1A2-Bca9011</span><span style='mso-tab-count:1'> </span>good</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Month="7" Day="29" Year="2006">Saturday, July 29, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The pBca9020-Bca9030 (2 clones, only clone 1 mapped and stocked) both gave a light pink pellet upon <span class=SpellE>pelleting</span> an LB w/o Mg culture.<span style='mso-spacerun:yes'> </span>#1 mapped right, so I don’t think I’ll characterize further.<span style='mso-spacerun:yes'> </span>The <span class=SpellE>sal</span> and Mg <span class=SpellE>NoB</span> subs both came out as weakened promoters, which is weird.<span style='mso-spacerun:yes'> </span>It’s almost as though the restriction sites pinned between the promoter and the ORF are terminators.<span style='mso-spacerun:yes'> </span>I might have to redesign that.<span style='mso-spacerun:yes'> </span>It definitely means I don’t want to do the Sal-<span class=SpellE>RfbC</span> splitting them up.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I did some mini and mapping:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1052"

type="#_x0000_t75" style='width:252pt;height:107.25pt'>
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</v:shape><![endif]--><![if !vml]><img width=336 height=143 src="Installment1-start072506_files/image056.jpg" v:shapes="_x0000_i1052"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pBca9012-1</span><span style='mso-tab-count:1'> </span>large <span class=SpellE>frag</span> too small</p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pBca9012-2</span><span style='mso-tab-count:1'> </span>large <span class=SpellE>frag</span> too small</p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pBca9012-3</span><span style='mso-tab-count:1'> </span>large <span class=SpellE>frag</span> too small</p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pBca9012-4</span><span style='mso-tab-count:1'> </span>correct (dominant phenotype, light pink)</p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pGLN-Bca1004</span><span style='mso-tab-count:1'> </span>good</p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pSB1A2-Bca9035</span><span style='mso-tab-count:1'> </span>good<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pSB1A2-Bca9023-1</span><span style='mso-tab-count:1'> </span>wrong<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pSB1A2-Bca9023-2</span><span style='mso-tab-count:1'> </span>wrong<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pSB1A2-Bca9023-3</span><span style='mso-tab-count:1'> </span>wrong</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The scheme for construction of pBca9012 was:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1053"

type="#_x0000_t75" style='width:6in;height:9pt'>
<v:imagedata src="Installment1-start072506_files/image057.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=576 height=12 src="Installment1-start072506_files/image058.gif" v:shapes="_x0000_i1053"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>That went very smoothly, got tons of small colonies in Ec100D:<span class=GramE>:pir</span>+.<span style='mso-spacerun:yes'> </span>At first they all looked white and I was worried.<span style='mso-spacerun:yes'> </span>A few were very red, and I grew clones 1-3 from those.<span style='mso-spacerun:yes'> </span>Clone 4 was a representative white colony.<span style='mso-spacerun:yes'> </span>Only clone 4 maps <span class=GramE>right,</span> and it was pink after saturation.<span style='mso-spacerun:yes'> </span>The plate colonies have all turned pink on the bench after a few days.<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I did <span class=SpellE>midipreps</span> of pSB1A2-J23012 (<span class=SpellE>SpR</span>), pSB1A2-Bca1010 (FRT), pSB1A2-Bca9008 (F-Cm-F), pBca1020-Bca1022-C2 (<span class=SpellE>Pcon</span>).<span style='mso-spacerun:yes'> </span>I’m digesting 23012 and 1010 and 9008 with <span class=SpellE>XbaI/PstI/CIP</span>.<span style='mso-spacerun:yes'> </span>I would do it with <span class=SpellE>AlwNI</span> instead of <span class=SpellE>PstI</span>, but we’re out.<span style='mso-spacerun:yes'> </span>I’m also doing 9008 with <span class=SpellE>HindIII/SpeI/CIP</span>.<span style='mso-spacerun:yes'> </span>Those are all being gel purified.<span style='mso-spacerun:yes'> </span>I purchased <span class=SpellE>oligo</span> CA1024F (<span class=SpellE>biotinylated</span> ca998) and did PCR ca1024F/G00101 on pSB1A2-Bca1011 mini with <span class=SpellE>Taq</span> on 200 <span class=SpellE>uL</span> scale and gel purified all of it.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I’m going to try solid-phase <span class=SpellE>biobrick</span> assembly with the above digests and some <span class=SpellE>BioMag</span> (Bangs labs) nuclease-free <span class=SpellE>streptavidin</span> beads.<span style='mso-spacerun:yes'> </span>These things are <span class=GramE>4.4 <span class=SpellE>ug</span>/mg biotin at 1.2 mg/<span class=SpellE>mL</span> for a binding capacity of 22 <span class=SpellE>uM</span> for biotin</span>.<span style='mso-spacerun:yes'> </span>So, 25 <span class=SpellE>uL</span> of beads has orders of magnitude more capacity than does 100 <span class=SpellE>ng</span> of a 500 <span class=SpellE>bp</span> DNA fragment capped in biotin.<span style='mso-spacerun:yes'> </span>That’s the volume I’ll start with for assembly.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1054"

type="#_x0000_t75" style='width:130.5pt;height:159.75pt'>
<v:imagedata src="Installment1-start072506_files/image059.emz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=174 height=213 src="Installment1-start072506_files/image060.gif" v:shapes="_x0000_i1054"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Since all the added cassettes are CIP treated, the <span class=SpellE>oly</span> 5’ phosphates are on the bead.<span style='mso-spacerun:yes'> </span>This should address the <span class=SpellE>stoichiometry</span> issues.<span style='mso-spacerun:yes'> </span>The last step will be to <span class=SpellE>ligate</span> on the SpeI-9008-HindIII plasmid cap, digest off the resin with <span class=SpellE>EcoRI</span> and <span class=SpellE>ligate</span> to circularize.<span style='mso-spacerun:yes'> </span>I might then hit half of it with T7 or T4 <span class=SpellE>pol</span> to fix the nicked strands, <span class=GramE>then</span> transform.<span style='mso-spacerun:yes'> </span>Some of the <span class=SpellE>midipreps</span> lost the pellet—I did this by the standard <span class=SpellE>HiSpeed</span> method which was stupid.<span style='mso-spacerun:yes'> </span>So, of the things I have to start with the best two constructs to try are a Spec-FRT part and a Spec-FRT-Spec part.<span style='mso-spacerun:yes'> </span>I think I’ll try both…we’ll see how I feel about that on Monday.<span style='mso-spacerun:yes'> </span>The little nicks due to CIP might become an issue with multiple parts.<span style='mso-spacerun:yes'> </span>Not sure.<span style='mso-spacerun:yes'> </span>All the nicks will be on one side of the DNA, so the thing can’t fall apart, but it might not transform well.<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I don’t know what’s up with 9023—I rechecked the sub scheme, and this guy really should be clean.<span style='mso-spacerun:yes'> </span>I think I’ll just redo it Monday—I must have just made an error somewhere.<span style='mso-spacerun:yes'> </span>I should probably start taking <span class=SpellE>pics</span> of the <span class=SpellE>subcloning</span> gels to be sure so I can look back at the source gels and know if anything was weird.<span style='mso-spacerun:yes'> </span>The colonies are white, and they map as the <span class=SpellE>wbbL</span> plasmid.<span style='mso-spacerun:yes'> </span>That’s the small <span class=SpellE>frag</span>, so it really should not have bled anything through.<span style='mso-spacerun:yes'> </span><span class=GramE>Hmmm.</span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Year="2006" Day="31" Month="7">Monday, July 31, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I’m doing the digests for the solid phase assembly:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1055"

type="#_x0000_t75" style='width:298.5pt;height:33.75pt'>
<v:imagedata src="Installment1-start072506_files/image061.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=398 height=45 src="Installment1-start072506_files/image062.gif" v:shapes="_x0000_i1055"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>All are looking good on the gel thus far.<span style='mso-spacerun:yes'> </span><span class=GramE>Doing a long gel to resolve the 1184/929 one.</span><span style='mso-spacerun:yes'> </span>Red is the desired band for each.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Meanwhile, I’ve started the beads.<span style='mso-spacerun:yes'> </span>I measured the amt of DNA in the <span class=SpellE>biotinylated</span> guy <span class=GramE>at around 75 <span class=SpellE>ng/uL</span></span>.<span style='mso-spacerun:yes'> </span>I’m doing 2 <span class=SpellE>uL</span> scale, but 2 of them, so I put 4 <span class=SpellE>uL</span> with 50 <span class=SpellE>uL</span> of beads.<span style='mso-spacerun:yes'> </span>The beads were washed 1x in 700 <span class=SpellE>uL</span> PBS, <span class=SpellE>resuspended</span> in 500 <span class=SpellE>uL</span> PBS, added the DNA, incubated 30 min, washed with water 1x, brought back up in 200 <span class=SpellE>uL</span> of 1X NEB2 + 1 <span class=SpellE>uL</span> <span class=SpellE>XbaI</span>.<span style='mso-spacerun:yes'> </span><span class=GramE>Digesting about 1 hr.</span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I did PCR mapping on 9020 and 9012:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1056"

type="#_x0000_t75" style='width:261pt;height:149.25pt'>
<v:imagedata src="Installment1-start072506_files/image063.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=348 height=199 src="Installment1-start072506_files/image064.jpg" v:shapes="_x0000_i1056"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1057"

type="#_x0000_t75" style='width:351pt;height:75pt'>
<v:imagedata src="Installment1-start072506_files/image065.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=468 height=100 src="Installment1-start072506_files/image066.gif" v:shapes="_x0000_i1057"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The pSB1A2-Bca9020 <span class=SpellE>PCRs</span> are perfect.<span style='mso-spacerun:yes'> </span>It confirms the location and presence of R6K, <span class=SpellE>CmR</span>, and <span class=SpellE>OriTr</span>.<span style='mso-spacerun:yes'> </span>It turns out I don’t have probes for FRT or <span class=SpellE>TnRev</span>, but the part junction is confirmed here.<span style='mso-spacerun:yes'> </span>I am proceeding with the <span class=SpellE>subclones</span>.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>On the pBca9012, the presence of <span class=SpellE>CmR</span>, <span class=SpellE>Tnp</span>, <span class=SpellE>OriT</span>, and R6K is confirmed…the sizes are all crazy.<span style='mso-spacerun:yes'> </span>I interpret this as being that the <span class=SpellE>frags</span> were too long for the ext time and I get <span class=GramE>a <span class=SpellE>mispriming</span></span> with 1002R.<span style='mso-spacerun:yes'> </span>The “C” band has the correct size and the <span class=SpellE>mispriming</span> size, but the rest are all <span class=SpellE>mispriming</span>, but they appear to all <span class=SpellE><span class=GramE>misprime</span></span> at the same two general regions.<span style='mso-spacerun:yes'> </span>I’m calling that good enough, will focus on getting a function confirmation here.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The status of the 1022 library is as follows.<span style='mso-spacerun:yes'> </span>Matt re-picked the following clones from the screen of 192 clones:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><span lang=PT-BR style='mso-ansi-language: PT-BR'>C2, D7, C5, H12, E1, B10, E11, F3, A5, F6, E10, C1, B9, F4, D7, C2, F12, A2, E8<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I grew up 6 clones (<span class=GramE>A</span><span lang=PT-BR style='font-family:Wingdings;mso-ascii-font-family: "Times New Roman";mso-hansi-font-family:"Times New Roman";mso-ansi-language: PT-BR;mso-char-type:symbol;mso-symbol-font-family:Wingdings'><span style='mso-char-type:symbol;mso-symbol-font-family:Wingdings'>à</span></span>F) of the ones that were “very red”. <span style='mso-spacerun:yes'> </span>Indeed they were very red at saturation, some more than <span class=SpellE>Ptet</span>.<span style='mso-spacerun:yes'> </span>The large <span class=GramE>set of clones that Matt picked were</span> well-isolated, so I am not <span class=SpellE>restreaking</span> those.<span style='mso-spacerun:yes'> </span>I did -80’s on those over the <span class=SpellE>wknd</span>, Matt is <span class=SpellE>miniprepping</span> today.<span style='mso-spacerun:yes'> </span>The <span class=GramE>A</span><span style='font-family:Wingdings;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-char-type:symbol;mso-symbol-font-family: Wingdings'><span style='mso-char-type:symbol;mso-symbol-font-family:Wingdings'>à</span></span>F guys were clearly mixed, so I <span class=SpellE>restreaked</span> those from the culture, picked more <span class=SpellE>cfu’s</span> yesterday.<span style='mso-spacerun:yes'> </span>Matt -80’d and <span class=SpellE>mini’d</span> those today.<span style='mso-spacerun:yes'> </span>All the <span class=SpellE>Tecan</span> data is in the file “072606-Tecan data…<span class=GramE>”:</span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1058"

type="#_x0000_t75" style='width:306pt;height:123pt'>
<v:imagedata src="Installment1-start072506_files/image067.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=408 height=164 src="Installment1-start072506_files/image068.gif" v:shapes="_x0000_i1058"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The data was mostly <span class=SpellE>consistant</span> with the first run, only a few changed <span class=GramE>position</span>.<span style='mso-spacerun:yes'> </span>The values on the A<span style='font-family:Wingdings;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-char-type:symbol;mso-symbol-font-family: Wingdings'><span style='mso-char-type:symbol;mso-symbol-font-family:Wingdings'>à</span></span>Fs is from the mixed culture, so those aren’t valid numbers.<span style='mso-spacerun:yes'> </span>I think the next step is to do some sequencing and see what we’re dealing with in terms of <span class=SpellE>miniprep</span> quality, degree of <span class=SpellE>cotransformed</span> hits, and sequence diversity.<span style='mso-spacerun:yes'> </span>Clearly I have a nice broad range of hits here.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Doing some <span class=SpellE>subcloning</span> today:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1059"

type="#_x0000_t75" style='width:6in;height:26.25pt'>
<v:imagedata src="Installment1-start072506_files/image069.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=576 height=35 src="Installment1-start072506_files/image070.gif" v:shapes="_x0000_i1059"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The 9023 is <span class=GramE>a re-do</span> from before.<span style='mso-spacerun:yes'> </span>Hopefully it will come out better this time.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>PCR with the 1023 20mers of irp9 with <span class=SpellE>Phusion</span> still looks like shit:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1060"

type="#_x0000_t75" style='width:80.25pt;height:129.75pt'>
<v:imagedata src="Installment1-start072506_files/image071.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=107 height=173 src="Installment1-start072506_files/image072.jpg" v:shapes="_x0000_i1060"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1061"

type="#_x0000_t75" style='width:431.25pt;height:10.5pt'>
<v:imagedata src="Installment1-start072506_files/image073.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=575 height=14 src="Installment1-start072506_files/image074.gif" v:shapes="_x0000_i1061"><![endif]><o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The right lane is a 1019 <span class=SpellE>pcr</span> off the 1023 <span class=SpellE>pcr</span>.<span style='mso-spacerun:yes'> </span>Not real sure where to go next there.<span style='mso-spacerun:yes'> </span>I think it’s some TA kit thing, though.<span style='mso-spacerun:yes'> </span>I think I’ll wait until my new Expand kit comes in. <span style='mso-spacerun:yes'> </span><span class=SpellE>Phusion</span> seems to generate a product even if there isn’t an appropriate template.<span style='mso-spacerun:yes'> </span>It primes too well.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The <span class=SpellE>PCRs</span> of the knockout with <span class=SpellE>Phusion</span> also looked shitty:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1062"

type="#_x0000_t75" style='width:82.5pt;height:110.25pt'>
<v:imagedata src="Installment1-start072506_files/image075.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=110 height=147 src="Installment1-start072506_files/image076.jpg" v:shapes="_x0000_i1062"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1063"

type="#_x0000_t75" style='width:245.25pt;height:22.5pt'>
<v:imagedata src="Installment1-start072506_files/image077.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=327 height=30 src="Installment1-start072506_files/image078.gif" v:shapes="_x0000_i1063"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Again, I think I’ll wait on the Expand kit.<span style='mso-spacerun:yes'> </span>The M band is the wrong size.<span style='mso-spacerun:yes'> </span>I’ll recheck that 279 <span class=GramE>is</span> the correct <span class=SpellE>oligo</span> to do <span class=SpellE>neuD</span>.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The Bca1025 AGGA mutant of pBca1021-E0040 (pBca1020-Bca1025) came out as about 100 <span class=SpellE>cfu</span>, all green.<span style='mso-spacerun:yes'> </span>I will redo that guy by overlap:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1064"

type="#_x0000_t75" style='width:431.25pt;height:29.25pt'>
<v:imagedata src="Installment1-start072506_files/image079.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=575 height=39 src="Installment1-start072506_files/image080.gif" v:shapes="_x0000_i1064"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>Having some difficulty on the solid phase assembly.</span><span style='mso-spacerun:yes'> </span>The digests all looked flawless.<span style='mso-spacerun:yes'> </span>The beads, however, appear to be sticking and/or degrading.<span style='mso-spacerun:yes'> </span>They don’t seem to be stable to multiple rounds of incubation in buffer.<span style='mso-spacerun:yes'> </span>Not sure how to remedy that.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I remapped the <span class=GramE>pSB1AK3-Bca9038’s</span> with <span class=SpellE>BamHI/PstI</span> and got the gel below.<span style='mso-spacerun:yes'> </span>Clone 1 is good, 2 is a dud.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1065"

type="#_x0000_t75" style='width:77.25pt;height:114.75pt'>
<v:imagedata src="Installment1-start072506_files/image081.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=103 height=153 src="Installment1-start072506_files/image082.jpg" v:shapes="_x0000_i1065"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Year="2006" Day="1" Month="8">Tuesday, August 01, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I did the solid phase assembly again today to make a FRT-Spec-FRT part in pSB1A2.<span style='mso-spacerun:yes'> </span>I first loaded 25uL of beads directly onto the pad of a <span class=SpellE>minelute</span> column.<span style='mso-spacerun:yes'> </span>The liquid immediate went into the column, so I added 200 <span class=SpellE>uL</span> of water and <span class=SpellE>pipetted</span> up and down to distribute it a little.<span style='mso-spacerun:yes'> </span>I added 100uL of 1X neb2, 1 <span class=SpellE>uL</span> <span class=SpellE>XbaI</span>, and 2.5 <span class=SpellE>uL</span> of the <span class=SpellE>biotinylated</span> PCR product.<span style='mso-spacerun:yes'> </span>Incubated 0.5 hour then did 100uL scale digestions and <span class=SpellE>ligations</span> with 1uL <span class=SpellE>SpeI</span> or 1uL <span class=SpellE>ligase</span> + 4uL <span class=SpellE>biobrick</span> part.<span style='mso-spacerun:yes'> </span>Did:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1066"

type="#_x0000_t75" style='width:176.25pt;height:186pt'>
<v:imagedata src="Installment1-start072506_files/image083.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=235 height=248 src="Installment1-start072506_files/image084.gif" v:shapes="_x0000_i1066"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Eluted the <span class=SpellE>qiaprep</span> in 10uL, setup <span class=SpellE>ligation</span> normally, transformed all of it, plated on Amp and Amp/Spec.<span style='mso-spacerun:yes'> </span>We’ll see what happens!</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The Bca1025 overlap PCR looked really good.<span style='mso-spacerun:yes'> </span>I <span class=SpellE>sub’d</span> <span class=SpellE>EcoRI/SpeI</span>, went directly into the pSB3C6 variation. <span style='mso-spacerun:yes'> </span>I <span class=GramE>won’t<span style='mso-spacerun:yes'> </span>be</span> able to confirm activity, but hopefully the <span class=SpellE>tRNA</span> that <span class=SpellE>Kaitlin</span> is making will all go well.<span style='mso-spacerun:yes'> </span>I might move the entire <span class=SpellE>tRNA</span> cassette into something just in case.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=MsoHyperlink><span style='color:windowtext;text-decoration:none;text-underline:none'>Bca9023, Bca9016, and Bca9017 all gave oodles of colonies, grew up 2 of each to screen tomorrow.<o:p></o:p></span></span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=MsoHyperlink><span style='color:windowtext;text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=MsoHyperlink><span style='color:windowtext;text-decoration:none;text-underline:none'>I setup new <span class=SpellE>PCRs</span> of pGLN-Bca1004 and pSB1A2-Bca9035 to do the <span class=SpellE>neuD</span> and <span class=SpellE>wbbL</span> <span class=SpellE>knockins</span>.<span style='mso-spacerun:yes'> </span><span class=GramE>Grew up Bos12/pKD46 from the -80 to do the comp.</span><o:p></o:p></span></span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=MsoHyperlink><span style='color:windowtext;text-decoration:none;text-underline:none'><o:p>&nbsp;</o:p></span></span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Month="8" Day="2" Year="2006">Wednesday, August 02, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1067"

type="#_x0000_t75" style='width:243pt;height:149.25pt'>
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</v:shape><![endif]--><![if !vml]><img width=324 height=199 src="Installment1-start072506_files/image086.jpg" v:shapes="_x0000_i1067"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The bands go:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1068"

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</v:shape><![endif]--><![if !vml]><img width=324 height=121 src="Installment1-start072506_files/image088.gif" v:shapes="_x0000_i1068"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>so</span>, looking good.<span style='mso-spacerun:yes'> </span>Stocking and saving the ones in red.<span style='mso-spacerun:yes'> </span>I <span class=SpellE>gp’d</span> the bands, doing the following <span class=SpellE>subclones</span>:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1069"

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</v:shape><![endif]--><![if !vml]><img width=575 height=31 src="Installment1-start072506_files/image090.gif" v:shapes="_x0000_i1069"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The subs all went smoothly, transformed TG1 or pir116 heat shock cells.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The F-Sp-F plates came out promising.<span style='mso-spacerun:yes'> </span><span class=GramE>Got about 200 <span class=SpellE>cfu</span> on Amp, about 15 on Amp/Spec.</span><span style='mso-spacerun:yes'> </span>I grew up 16 from amp and 8 from Asp to screen by colony <span class=SpellE>pcr</span> tomorrow.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The <span class=SpellE>neuD</span> and <span class=SpellE>wbbL</span> <span class=SpellE>knockin</span> <span class=SpellE>pcrs</span> came out good with expand, so I <span class=SpellE>dig’d</span> with <span class=SpellE>DpnI</span> and transformed Bos12/pKD46.<span style='mso-spacerun:yes'> </span>Hopefully we’ll have clones tomorrow!</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The pSB3C6-Bca1025 plate gave lots of <span class=SpellE>cfus</span>, none were green, grew up 2 to screen.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Month="8" Day="3" Year="2006">Thursday, August 03, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I got no colonies for the bos12 knockouts.<span style='mso-spacerun:yes'> </span><span class=GramE>Will repeat tomorrow at higher OD.</span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I <span class=SpellE>mini’d</span> and passed off the Bca1025’s to <span class=SpellE>Kaitlin</span> for her to stock and sequence.<span style='mso-spacerun:yes'> </span>I’ll put the construction file up tonight.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The 9039 and NES subs looked great.<span style='mso-spacerun:yes'> </span>Light pink for <span class=SpellE>pBACr</span>-NES, grew up 1.<span style='mso-spacerun:yes'> </span>Grew 2 of the <span class=GramE>pSB1AK3-Bca9039’s</span>.<span style='mso-spacerun:yes'> </span>Tomorrow I will put the <span class=SpellE>wbbL</span> and <span class=SpellE>rfbC</span> cassettes into <span class=SpellE>pBACr</span>-NES and also blunt out the <span class=SpellE>XbaI</span> site for <span class=SpellE>pBACr-NESd</span>.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Adam <span class=SpellE>Deutschbauer’s</span> dap variant of BW20767 (WM3064 I think) grew fine on the plate, grew up a colony to do comp tomorrow.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The pBca9045 and pBca9046 gave <span class=SpellE>shitloads</span> of small colonies.<span style='mso-spacerun:yes'> </span>I grew 2 of each.<span style='mso-spacerun:yes'> </span>I probably should have done a <span class=SpellE>neg</span> on the cells, it looked a little sketchy, but hopefully correct.<span style='mso-spacerun:yes'> </span>Those are in pir116, so they might be sickly.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The solid phase assembly of FRT-Spec-FRT looks funny.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1070"

type="#_x0000_t75" style='width:6in;height:138.75pt'>
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</v:shape><![endif]--><![if !vml]><img width=576 height=185 src="Installment1-start072506_files/image092.jpg" v:shapes="_x0000_i1070"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The gel is of 24 clones as <span class=SpellE>Taq</span> G00101/ca998 colony PCR.<span style='mso-spacerun:yes'> </span>None are obviously correct.<span style='mso-spacerun:yes'> </span>The markers are the <span class=GramE>1kb,</span> I’d expect something like 1.1kb. <span style='mso-spacerun:yes'> </span>The 16 on the left were <span class=GramE>naïve,</span> the 8 on the right were <span class=SpellE>specR</span>. <span style='mso-spacerun:yes'> </span>I don’t really need the construct, so I think I’ll just move on to a new strategy—doing it on plates like an <span class=SpellE>elisa</span>.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>Still having trouble cloning <span class=SpellE>salS</span>.</span><span style='mso-spacerun:yes'> </span>Not sure I really want to put more effort into this guy.<span style='mso-spacerun:yes'> </span>I did 1023F/R <span class=SpellE>pcr</span> with Expand on Y <span class=SpellE>pseud</span>. gen.<span style='mso-spacerun:yes'> </span><span class=GramE>Got multiple bright bands.</span><span style='mso-spacerun:yes'> </span>1019F/R <span class=SpellE>pcr</span> on that gave no band.<span style='mso-spacerun:yes'> </span>I setup 1019F/1023R and 1023F/1019R to see if it’s just one of the 1019’s that fails.<span style='mso-spacerun:yes'> </span>If so, I’ll just TA it and go from there.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I also <span class=SpellE>sub’d</span> the 901F/R <span class=SpellE>mgrB</span> <span class=SpellE>pcr</span> into pBAC583 (old <span class=SpellE>NoB</span> dig).<span style='mso-spacerun:yes'> </span>Trans TG1.<span style='mso-spacerun:yes'> </span>The purpose of this is to revisit the pir116/R6K regulation library.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Year="2006" Day="4" Month="8">Friday, August 04, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I did some mapping:<span style='mso-spacerun:yes'> </span>first lane is <span class=SpellE>pBACr</span>-NES, second two are pSB1AK3-Bca9039, <span class=GramE>all</span> are <span class=SpellE>EcoRI/SpeI</span> digs.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1071"

type="#_x0000_t75" style='width:135pt;height:132.75pt'>
<v:imagedata src="Installment1-start072506_files/image093.gif" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=180 height=177 src="Installment1-start072506_files/image093.gif" v:shapes="_x0000_i1071"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>Looks good, going with 9039 clone 1.</span><span style='mso-spacerun:yes'> </span>I made -80’s of those and WM3064, then <span class=SpellE>subcloned</span> 9037<span class=GramE>,9038</span>, and 9039 with ES into <span class=SpellE>pBACr</span>-NES.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The 9045 and 9046 guys are clearly massively contaminated.<span style='mso-spacerun:yes'> </span>The lawn is that funny yellow junk.<span style='mso-spacerun:yes'> </span>There are a few pink guys in there, I picked one from each plate and <span class=SpellE>restreaked</span> it today.<span style='mso-spacerun:yes'> </span><span class=GramE>Will grow them up tomorrow, hopefully.</span><span style='mso-spacerun:yes'> </span>For the BAC transformations I’m using the same cells but doing Kan/Tri, and hopefully that will kill the garbage.<span style='mso-spacerun:yes'> </span>I’m also running <span class=SpellE>negs</span> on K and C for the cells to see what’s up.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>There were some colonies on the <span class=SpellE>neuD/wbbL</span> <span class=SpellE>knockins</span>, so I just put the bos12/pKD46 in the fridge and grew up the colonies.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pBAC901</span> looked solid.<span style='mso-spacerun:yes'> </span>~0.5% strong green <span class=SpellE>cfus</span>, rest were light green.<span style='mso-spacerun:yes'> </span><span class=GramE>Picked a light green.</span><span style='mso-spacerun:yes'> </span>Will mini and sub the pir116/R6K lib tomorrow (if I can find it).</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The <span class=SpellE>SalS</span> <span class=SpellE>PCRs</span> failed again.<span style='mso-spacerun:yes'> </span>I think I’ll just drop that…not worth the trouble.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I did a standard Vent blunting of <span class=SpellE>pBACr</span>-NES to make <span class=SpellE>pBACr-NESd</span>.<span style='mso-spacerun:yes'> </span>This guy has everything <span class=SpellE>biobrick</span>-unique except <span class=SpellE>PstI</span>.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Month="8" Day="7" Year="2006">Monday, August 07, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I grew up and <span class=SpellE>gen</span> prepped the one colony of the <span class=SpellE>neuD</span> <span class=SpellE>knockin</span> (L1) and two of the <span class=SpellE>wbbL</span> <span class=SpellE>knockins</span> (M1 and M2).<span style='mso-spacerun:yes'> </span>I did <span class=SpellE>PCRs</span> and <span class=SpellE>EcoRI</span> digestion to map:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1072"

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</v:shape><![endif]--><![if !vml]><img width=443 height=45 src="Installment1-start072506_files/image095.gif" v:shapes="_x0000_i1072"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1073"

type="#_x0000_t75" style='width:117pt;height:90.75pt'>
<v:imagedata src="Installment1-start072506_files/image096.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=156 height=121 src="Installment1-start072506_files/image097.jpg" v:shapes="_x0000_i1073"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The two <span class=SpellE>wbbL</span> <span class=SpellE>knockins</span> look <span class=GramE>good,</span> <span class=SpellE>neuD</span> looks like it may be a dud…maybe.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Did some mini and mapping:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1074"

type="#_x0000_t75" style='width:225pt;height:117.75pt'>
<v:imagedata src="Installment1-start072506_files/image098.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=300 height=157 src="Installment1-start072506_files/image099.jpg" v:shapes="_x0000_i1074"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>pBca9045</p>

<p class=MsoNormal style='tab-stops:135.0pt'>pBca9046</p>

<p class=MsoNormal style='tab-stops:135.0pt'>Bca9037-1</p>

<p class=MsoNormal style='tab-stops:135.0pt'>Bca9037-2</p>

<p class=MsoNormal style='tab-stops:135.0pt'>Bca9038-1</p>

<p class=MsoNormal style='tab-stops:135.0pt'>Bca9038-2</p>

<p class=MsoNormal style='tab-stops:135.0pt'>Bca9039-1</p>

<p class=MsoNormal style='tab-stops:135.0pt'>Bca9039-2</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Year="2006" Day="10" Month="8">Thursday, August 10, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I <span class=SpellE>mini’d</span> and mapped 2 of 9040, 9041, and 9042, the <span class=SpellE>pBACr</span>- <span class=SpellE>Piptg-wbbL</span> and <span class=SpellE>Psal-rfbC’s</span>.<span style='mso-spacerun:yes'> </span>Looks a little weird (<span class=SpellE>XbaI</span> digs):</p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1075"

type="#_x0000_t75" style='width:183.75pt;height:95.25pt'>
<v:imagedata src="Installment1-start072506_files/image100.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img width=245 height=127 src="Installment1-start072506_files/image101.jpg" v:shapes="_x0000_i1075"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>It’s all too big.<span style='mso-spacerun:yes'> </span>I think I need some PCR mapping to see if they are in the ballpark or way off.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The mgrB/pir-r6K business gave a lawn of bacteria.<span style='mso-spacerun:yes'> </span>I scraped the <span class=GramE>plate,</span> <span class=SpellE>mini’d</span>, did 901F/875R PCR and got out a 1kb band—too small.<span style='mso-spacerun:yes'> </span>I have concerns about pBAC901—it wasn’t green enough in the pellet and there were some faint additional bands in the digest.<span style='mso-spacerun:yes'> </span>Something’s up with that guy.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Month="8" Day="15" Year="2006">Tuesday, August 15, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I did some PCR mapping of the pBACr-Bca9040, Bca9041, and Bca9042 with ca605F/ca606R.<span style='mso-spacerun:yes'> </span>I got no product but did using <span class=SpellE>pBACr</span>-NES as a control and it gave a good product.<span style='mso-spacerun:yes'> </span>So, I think the pir116 is still having contamination issues.<span style='mso-spacerun:yes'> </span>The mapping of the pBca9045 and pBca9046 also came out <span class=GramE>weird,</span> I think it is all just contamination in that strain.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Today I re-<span class=SpellE>ligated</span> pBca9045 and pBca9046 and transformed directly into BW20767.<span style='mso-spacerun:yes'> </span>Hopefully that will work better—these are the cells the kids made, and they transformed pBca9012 pretty cleanly (almost all red colonies, I grew up one did a -80 today).</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I did a small scale comp of pSB3C6-Bca1025 (the GFP-AGGA guy) and transformed in <span class=SpellE>Kaitlin’s</span> two <span class=SpellE>tRNA</span> constructs.<span style='mso-spacerun:yes'> </span>I got tons of <span class=SpellE>cfus</span>, but I didn’t do a <span class=SpellE>neg</span> control.<span style='mso-spacerun:yes'> </span>Suffice it to say, they weren’t green on the plate or upon growing to <span class=SpellE>sat’n</span>.<span style='mso-spacerun:yes'> </span>I’m not sure if this is just a contaminated prep (seems unlikely) or no activity in either the reporter or the <span class=SpellE>tRNA</span>.<span style='mso-spacerun:yes'> </span>My plan is to move the Bca1025 reporter back up to pSB1A2 and put it with the original Ser2-AGGA reporter.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I ordered a new <span class=SpellE>oligo</span> for doing pir116:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><span lang=PT-BR style='mso-ansi-language: PT-BR'>ca1026R<span style='mso-tab-count:1'> </span>Reverse oligo for pir116<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span lang=PT-BR style='mso-ansi-language: PT-BR'>CCATAgaattcGCCATATATCACCCCTTAGC<o:p></o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span lang=PT-BR style='mso-ansi-language: PT-BR'><o:p>&nbsp;</o:p></span></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The <span class=SpellE>pcr</span> went fine, <span class=SpellE>ligated</span> into pBACr899 and pBACr-mgr901. <span style='mso-spacerun:yes'> </span>I got no colonies on <span class=SpellE>SalK</span> for the 899 variants and all green colonies on the 901 plate.<span style='mso-spacerun:yes'> </span>Not sure why that’s going wrong.<span style='mso-spacerun:yes'> </span>I’m suspecting that either there’s a toxicity thing or the <span class=SpellE>BamHI</span> site on the 977F <span class=SpellE>oligo</span> is screwed up.<span style='mso-spacerun:yes'> </span>I’m going to take an alternate route here:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>PCR ca56/ca1026R on pBAC905<span style='mso-tab-count:1'> </span>(<span class=SpellE>bp</span>, <span class=SpellE>BamHI/EcoRI</span>)<o:p></o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>Sub into pBACr-Mgr901<span style='mso-tab-count:1'> </span><span style='mso-tab-count:2'> </span>(<span class=SpellE>BamHI/EcoRI</span>)</p>

<div style='border:none;border-bottom:solid windowtext 1.0pt;mso-border-bottom-alt: solid windowtext .75pt;padding:0in 0in 1.0pt 0in'>

<p class=MsoNormal style='tab-stops:135.0pt;border:none;mso-border-bottom-alt: solid windowtext .75pt;padding:0in;mso-padding-alt:0in 0in 1.0pt 0in'>Product is pBACr-MgrPir56</p>

</div>

<p class=MsoNormal style='tab-stops:135.0pt'>EIPCR ca901R/ca977F on MgrPir56<span style='mso-tab-count:1'> </span>(<span class=SpellE>bp</span>, <span class=SpellE>BamHI</span>/Vent)</p>

<p class=MsoNormal style='tab-stops:135.0pt'>Product is pBACr-MgrPir977 lib</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>That should take care of any restriction site issues.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Year="2006" Day="17" Month="8">Thursday, August 17, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'>All the subs yesterday went well… </p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>pBACr-MgrPir56</span> (described above, got about 100 <span class=SpellE>cfu</span>, grew up 3, in TG1)</p>

<p class=MsoNormal style='tab-stops:135.0pt'>Bca1025 into <span class=GramE>pSB1AK3(</span>-b0015) (about 500 <span class=SpellE>cfu</span> in TG1, grew up 2), it did this because the <span class=SpellE>tRNAs</span> weren’t green.<span style='mso-spacerun:yes'> </span><span class=GramE>Going to put this new reporter with the old pAC-Ser2AGGA to test the reporter.</span></p>

<p class=MsoNormal style='tab-stops:135.0pt'>pBACr-90* (40,41,42) <span class=SpellE>resub’d</span> into <span class=SpellE>pBACr-NESd</span> the original scheme, transformed into BW20767, got ~50 <span class=SpellE>cfu</span> of each, looked much more normal.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I <span class=SpellE>religated</span> the pBca9045/9046 the other day, transformed directly into BW20767.<span style='mso-spacerun:yes'> </span>The 9046’s are decently red, possibly full on.<span style='mso-spacerun:yes'> </span>Cell pellets of 9045 are pinkish, colonies are white.<span style='mso-spacerun:yes'> </span>I did <span class=SpellE>triparental</span> mating with two individual clones of these guys with pBca9012/BW and RU1012 as the recipient, plating on KC.<span style='mso-spacerun:yes'> </span>I got dusty lawns, looks like transposition, though, for the ones where 9045 or 9046 was added.<span style='mso-spacerun:yes'> </span>I should have added <span class=SpellE>aTc</span> to these, but forgot.<span style='mso-spacerun:yes'> </span>It all seems to work, though.<span style='mso-spacerun:yes'> </span>I’ve <span class=SpellE>replated</span> MC600u, going to transform pBACr-UG784 (<span class=SpellE>Ptet-uppgen</span>), show first that those are <span class=SpellE>genR<span class=GramE>,uppS</span></span>, then do transposition and see if I can find some interesting promoters with that.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'>I’m getting some P1vir from the <span class=SpellE>Bustamante</span> lab <span class=GramE>tomorrow,</span> the protocol for transduction is here:</p>

<p class=MsoNormal style='tab-stops:135.0pt'><a href="http://www.openwetware.org/wiki/Sauer:P1vir_phage_transduction">http://www.openwetware.org/wiki/Sauer:P1vir_phage_transduction</a></p>

<p class=MsoNormal style='tab-stops:135.0pt'><span class=GramE>Going to try that on the <span class=SpellE>knockins</span> of Bos12 for <span class=SpellE>wbbL</span> and <span class=SpellE>neuD</span>.</span><span style='mso-spacerun:yes'> </span>Going to both try to transfer the cassettes back to Bos12 and pass them to MG1655 and MC1061.<span style='mso-spacerun:yes'> </span>It would be really nice if that all could work in MC1061.<span style='mso-spacerun:yes'> </span>We’ll see.</p>

<p class=MsoNormal style='tab-stops:135.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:135.0pt'><st1:date Month="8" Day="18" Year="2006">Friday, August 18, 2006</st1:date></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1076"

type="#_x0000_t75" style='width:252pt;height:105.75pt'>
<v:imagedata src="Installment1-start072506_files/image102.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img border=0 width=336 height=141 src="Installment1-start072506_files/image103.jpg" v:shapes="_x0000_i1076"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1077"

type="#_x0000_t75" style='width:225pt;height:75.75pt'>
<v:imagedata src="Installment1-start072506_files/image104.png" o:title=""/>

</v:shape><![endif]--><![if !vml]><img border=0 width=300 height=101 src="Installment1-start072506_files/image105.jpg" v:shapes="_x0000_i1077"><![endif]></p>

<p class=MsoNormal style='tab-stops:135.0pt'>The above gel(s) goes:</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>ES<span style='mso-tab-count:1'> </span>pBACr-9040-1</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>ES<span style='mso-tab-count:1'> </span>pBACr-9040-2</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>ES<span style='mso-tab-count:1'> </span>pBACr-9041-1</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>ES<span style='mso-tab-count:1'> </span>pBACr-9041-2</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>ES<span style='mso-tab-count:1'> </span>pBACr-9042-1</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>ES<span style='mso-tab-count:1'> </span>pBACr-9042-2</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>ES<span style='mso-tab-count:1'> </span>pSB1AK3-Bca1025-1</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>ES<span style='mso-tab-count:1'> </span>pSB1AK3-Bca1025-2</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>BE<span style='mso-tab-count:1'> </span>pBACr-MgrPir56-1</p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-tab-count:1'> </span>BE<span style='mso-tab-count:1'> </span>pBACr-AraT7940-F11 from pir116 for <span class=SpellE>voigt</span> stock</p>

<p class=MsoNormal style='tab-stops:81.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:81.0pt'>So, all look good except pSB1AK3-Bca1025-2.<span style='mso-spacerun:yes'> </span>Did -80s of all the clone 1’s and saved minis.<span style='mso-spacerun:yes'> </span>I transformed MG1655 or MG/pMF19 with the <span class=SpellE>relavent</span> pBACr904*’s.<span style='mso-spacerun:yes'> </span>I’m going to do the P1 transduction business on several guys and do all the results together on one O-gel.</p>

<p class=MsoNormal style='tab-stops:81.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:81.0pt'>On the <span class=SpellE>neuD/wbbL</span> <span class=SpellE>knockins</span>, I had another colony on the “L” plate (the <span class=SpellE>neuD</span> <span class=SpellE>knockin</span>).<span style='mso-spacerun:yes'> </span>I grew that up yesterday.<span style='mso-spacerun:yes'> </span>It <span class=SpellE>gen</span>-prepped like an E. coli, and I setup the analytical PCR:</p>

<p class=MsoNormal style='tab-stops:81.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1078"

type="#_x0000_t75" style='width:332.25pt;height:12pt'>
<v:imagedata src="Installment1-start072506_files/image106.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img border=0 width=443 height=16 src="Installment1-start072506_files/image107.gif" v:shapes="_x0000_i1078"><![endif]><o:p></o:p></p>

<p class=MsoNormal style='tab-stops:81.0pt'><span style='mso-spacerun:yes'> </span>We’ll see how that looks tomorrow.<span style='mso-spacerun:yes'> </span>I did an amplification of the <span class=SpellE>Bustamante</span> P1 stock.<span style='mso-spacerun:yes'> </span>It totally cleared my culture of MC600u.<span style='mso-spacerun:yes'> </span>So, that’s good.<span style='mso-spacerun:yes'> </span>I’ll do the transduction tomorrow and try sending things into MG1655, Bos12, and MC600u.<span style='mso-spacerun:yes'> </span>So, I grew up cultures of all that today.</p>

<p class=MsoNormal style='tab-stops:81.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:81.0pt'>I also transformed MC600u with pBACr-UG784.<span style='mso-spacerun:yes'> </span>I’ll do the first <span class=SpellE>transposon</span> library on that.</p>

<p class=MsoNormal style='tab-stops:81.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:81.0pt'>Since the pBACr-MgrPir56 looks pretty good, the mini clearly shows higher copy even though it’s in TG1, so it must be working to some degree.<span style='mso-spacerun:yes'> </span>I setup a PCR:</p>

<p class=MsoNormal style='tab-stops:81.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1079"

type="#_x0000_t75" style='width:6in;height:10.5pt'>
<v:imagedata src="Installment1-start072506_files/image108.wmz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img border=0 width=576 height=14 src="Installment1-start072506_files/image109.gif" v:shapes="_x0000_i1079"><![endif]></p>

<p class=MsoNormal style='tab-stops:81.0pt'>That will give a plasmid that looks like:</p>

<p class=MsoNormal style='tab-stops:81.0pt'><!--[if gte vml 1]><v:shape id="_x0000_i1080"

type="#_x0000_t75" style='width:126pt;height:90.75pt'>
<v:imagedata src="Installment1-start072506_files/image110.emz" o:title=""/>

</v:shape><![endif]--><![if !vml]><img border=0 width=168 height=121 src="Installment1-start072506_files/image111.gif" v:shapes="_x0000_i1080"><![endif]></p>

<p class=MsoNormal style='tab-stops:81.0pt'>If I do the closure of the circle with <span class=SpellE>NotI</span>, the site will be destroyed.<span style='mso-spacerun:yes'> </span>I can also close it up with <span class=SpellE>BglII</span> partial digestion.<span style='mso-spacerun:yes'> </span>I haven’t decided yet which is better.<span style='mso-spacerun:yes'> </span>The <span class=SpellE>gameplan</span> is to do this, make sure the thing replicates, <span class=GramE>then</span> make the EIPCR library directly on it to make the Mg-sensitive <span class=SpellE>replicon</span> (if this guy isn’t already Mg-sensitive).</p>

<p class=MsoNormal style='tab-stops:81.0pt'><o:p>&nbsp;</o:p></p>

<p class=MsoNormal style='tab-stops:81.0pt'>The <span class=SpellE>riboregulator</span> stuff is looking really good, so I don’t think we need 4-base anymore.<span style='mso-spacerun:yes'> </span>I have the Bca1025 in case we need to revisit it.<span style='mso-spacerun:yes'> </span>If I revisit all that, this guy needs sequencing (it was a sloppy <span class=SpellE>subclone</span>) and then can put in pAC-Ser2AGGA to confirm activity.<span style='mso-spacerun:yes'> </span><span class=GramE>If that works, can revisit the <span class=SpellE>biobricked</span> four-base.</span><o:p></o:p></p>

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