July entries

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Amy N. Kristofferson 14:21, 29 July 2010 (EDT)
To do:
 * MP 41/42 stuff.
 * Map minipreps
 * Submit for sequencing
 * Pick colonies off 9145-1144

Background:
 * Look into adding his-tags to transposases for purification
 * Look into buffers that optimize transposase activity

iGEM10_046 and 051 Assembly analysis
ColPCR



lanes 1-8: 42+44 (iGEM10_046) expected length: 6317bp  Perfect: 4,7  MP: 4,7

lanes 9-12: 41+43 (iGEM10_049) expected length: 5978bp  Perfect: 4   MP: 1,2 (4 was cotransformed)

Cotransformation results: Only 41+43d was cotransformed. Unfortunately, d was the only one that looked good on the colPCR. Oh well.

Miniprepped: 42+44: c,d,g (labeled 46c,d,g for iGEM10_046) 41+43: a,b,c (labeled 49a,b,c for iGEM10_049)

Assembly of 1888+1858 with Mike's plasmid
Messed up yesterday's assembly, and realized I don't need to gel purify the digest of Mike's plasmid, because I'll be able to select for the correct plasmids, since the colonies will be green So I set up the following:

Bgl/XhoI digest of 1884+1858 e,f eluted PCR products

Bam/XhoI digest of Mike's plasmid (mel001 in pJco12)

Started at 12:55pm.

Ligation started at 2:20pm.

Transformed jtk030 cells and plated on amp.

Amy N. Kristofferson 14:10, 28 July 2010 (EDT)
To do:
 * Pick colonies off of 41/42+43/44 plates
 * Pick colonies for Tecan assay tomorrow (see Tim's email)

41/42+43/44 plates Analysis and colony picking
42+44: 18 colonies 42+43: 0 colonies 41+44: 0 colonies 41+43: tons of colonies

I'll pick 8 colonies off 42+44 (I have a feeling a lot of them are co-transformed), and 4 colonies off 41+43. Inoculate in LB-AK, colPCR?, streak on triple antibiotics.

Ran a 7KcolPCR

Restock 9145-1144
Transformed jtk030 cells.

1884+1858 ca998/iGEM048
Expected length: ~700bp. They all look great!



I'll also digest Mike's plasmid (Mel001 p3co12) with Bam/XhoI. Started at 2:14pm.

Even though I haven't gotten sequencing results back, I'm going to digest sample c and d with Bgl/XhoI. Digest started at 3:06pm. Gel purified. Expected lengths: 700bp dropout, vector should be ~5700bp. (Single cut would produce ~6400bp) Hopefully the gp worked well... I cut out the upper band, which was too high to really distinguish the length.

Ligation started at 5:29pm.

Transformed jtk030 cells.

Found my digests in the incubator. Turns out I used the undigested eluted PCR product for the assembly. Whoops. Trashed the cells. I'll redo the digestion+ligation tomorrow.

Transposase Assay: Testing Transposase Expression time and Activity Time

 * 1) Day before, add one colony to 6mL of LB-CK.
 * 2) Day of assay, move 1000uL of cells into five flasks (can discard extra mL)
 * 3) Add 10uL of arabinose for each mL of cells at t=o, 1 hour, 2 hours, 3 hours
 * 4) t=0 was 10:58am. I added 10ul of ara to the tube marked t=0.
 * 5) t=1 was 11:56am. I added 10uL of ara to the tube marked t-1.
 * 6) t=2 was 12:58pm.
 * 7) t=3 was 1:58pm.


 * 1) After four hours have passed since you first added arabinose (3pm), add 1uL of atc per mL of cells. Allow 1 hour for lysis.
 * 2) Added 1uL of atc to every tube but the control at 3:00pm.
 * 3) After that one hour, add 3uL of 9145-1144 DNA to each tube.
 * 4) Added DNA at 4:00pm. There was no visible sign that the cells had lysed.
 * 5) Remove a 200uL sample of lysate after 30min, 1 hour, 1.5hour, 2hours.
 * 4:30, 5, 5:30, 6pm (NOTE: I may have forgotten to add ADB to the lysate for the 5:00pm sample... I can't remember)
 * 1) Immediately after removal, spin down the lysate and zymo the supernatant.
 * 2) Transform lefty pir+ cells with the lysate. Plate on Amp and Gen.

Need to transform cells w/ 9145-1144 so we can get more DNA.

Transformed TG1 cells.

Rough test of Self Lysis Timing in TB
Yesterday, I inoculated iGEM10_047 and 048. I put 1 mL of each into two tubes, so I have a control tube and a tube to add atc to, with all the cells derived from one colony pick.

Added 1uL of Conor's 1000x atc at 11:25am. At 12:25pm, the cells still do not appear to have lysed.

Check cells at 11:55am. Based on visual inspection, there appears to be no change in OD.

Checked cells again at 1:00pm. The cells in 47 have clearly lysed, but it doesn't appear that the 48 cells have lysed yet.

Pictures: 47 (w/o tip) Left=control, right=atc 48 (w/ tip) left=control, right=atc.

Sequencing Results
iGEM10_042a is perfect. (ca998 sequencing redo produced a perfect partial)

Degradation Assay
Conor will do this tomorrow:

Add 1uL of atc to each 1mL sample of cells. Let cells lyse for an hour. Add 1uL of DNA. At each of the following intervals: 15min, 30min, 45min, 1 hour, 1.5, 2, 2.5, 3, remove 200uL of DNA, spin down, and zymo supernatant. After you have all your zymo'd samples, plate on Amp.

Amy N. Kristofferson 14:25, 27 July 2010 (EDT)
To do:
 * Grow up Payload delivery colonies in TB
 * Grow up transposase colonies

Picked two colonies of jtk030 cells transformed with iGEM10_047 and 048 and put them in TB-KA in the shaker upstairs. Tomorrow, I'll see how long it takes for the cells to undergo self-lysis. Each part has the exact same SL device under the same Ptet promoter, so they shouldn't vary.

PCR off 1884+1858 in 1601CK
I designed an oligo that will add a XhoI site to the NLS. So if I do a PCR with ca998 and my oligo igemTen048, hopefully I should get a band around 733bp long. I should only get this band if NLS is after RFP. It can't PCR off just RFP, because there would be no NLS to bind to. And just NLS is only 33bp, so that wouldn't even show up on the gel. Ran a 2K55.

Mapping of 1884 and 1858
I wasn't going to map these, but since sequencing of 1884+1858 a and b w/ g00101 both failed, I'm going to map them. The maps won't show whether or not the NLS tag is there, but it should show how good the MP is and if the backbone is good.

Eco/Bam Digest started at: 12:30pm

Expected lengths: 3142 (vector), 711 (part)

Lanes 1-8 correspond with samples a-h.



Everything looks good. They could have had bad reads because they were cotransformed. I submitted 1884+1858c and d for sequencing.

Sequencing Results
iGEM10_020b in pmllCA is perfect iGEM10_042a in pmllAC's fwd read was messed up. I submitted the exact same part w/ ca998 again. iGEM10_041b in pmllCA is perfect

Next Assembly step: Creating iGEM10_045/046 and 049/050 (Self-Lysis T ver)
Assemble iGEM10_042/041 with iGEM10_43/044

I have left over righty digest of iGEM10_043/044, so I only have to set up a Lefty digest of iGEM10_042/041. NOTE: iGEM10_042 hasn't been sequence confirmed yet, but I'm going to go ahead with assembly and keep my fingers crossed.

I think I started it around 2:00pm.

Started ligation at 4:37pm. Used 2114+2275/2316 digests from construction of 47 and 48.

Amy N. Kristofferson 14:07, 26 July 2010 (EDT)
To do:
 * Transform cells w/ best igem20 map
 * order oligos
 * submit best igem20,41,42 map for sequencing
 * start next assembly step for 41, 42?

Oligos
Designed the following oligo: igemTen048     gctagCTCGAGttaAACTTTACGTTTTTTTTTCGGCGG  38      rev xhoI 1858 {<NLS!}

ColPCR results


Expected length: about 900bp. They all look good. Mapping won't show me much, since the NLS is so small (~30bp). So I'm going to sumbit two samples for sequencing, using the g00101 oligo. If they're cotransformed, I'll get back a mixed read.



Expected lengths for iGEM10_042: 2895bp (lanes 1-4) and iGEM10_041: 3126bp (lanes 5-8)

Results: 42a,b,c and 41b,d look good. I'll map all the minipreps anyway.

Miniprep Mapping
They all look good! iGEM10_020 is a little faint, but I'm going to send off a sample of each to be sequenced.



I ran the same gel out for a few more minutes... You can see the sizes of the 42 and 41 digests better now...



started at 12:00pm.

Transformed jtk030 with iGEM10_020b
Started rescue at 2:25pm. Labeled "Sequenced MP of iGEM10_020b." I'll grow up colonies tomorrow so I can perform the Transposase Assay on Wednesday.

Sequencing submissions
Submitted iGEM10_020b in pmllCA, iGEM10_042a in pmllAC, iGEM10_041b in pmllCA, and jh1884+1858 in 1601CK A,B for sequencing.

Bam/XhoI digest started at 1:50pm.

Amy N. Kristofferson 14:07, 24 July 2010 (EDT)
To do:
 * Run gel of colPCRs from Friday and Sat.
 * Map Minipreps of 1884+1848.
 * Miniprep any iGEM10_020's that aren't co-transformed.
 * Miniprep the 41/42 samples w/ good colPCR results.
 * figure out what to do w/ ef1a and atub

Colony picking: 42/41PCR+pMLL4AC and iGEM10_020
Picked 8 colonies of iGEM10_020 and checked for cotransformation. Didn't run a colPCR b/c the part is too big. Picked 4 colonies of 42/41PCR+pMLL4AC and started a 4K colPCR. Had to make more colPCR MM.

Miniprepped all 1884+1858 samples
Labelled 1884+1858a-h and placed in Amy #2. I need to map them.

Sequencing Results for iGEM10_019b
The part looks great! I'm glad I continued on with assembly.

Amy N. Kristofferson 13:45, 23 July 2010 (EDT)
To do:
 * analytical gel of PCR of 42+41
 * if it looks good, digest eco/bam and ligate w/ pMLL4AC.
 * MP iGEM10_019b,d
 * Map
 * Submit for sequencing
 * Start next stage of assembly
 * Pick colonies from 1884, 1858. ColPCR, innoculate. No need to check for co-transf (same color)Tomorrow, miniprep and eco/bam into 9145. (xhoI site right after Bam)
 * Pick lots of colonies from ef1a/atub+2093 plate.
 * Redo-transformation of 47 and 48 (nothing grew on Tahoura's plates)

Later...
 * Pick colonies of igem47,48. Rough test to see how long it takes bacteria to lyse in TB

Colony Picks
1884+1858: expected colPCR length: 912bp efla/atub+2093 expected colPCR lengths: 1952, 2190bp

Ran col3k pcr.

Assembly: iGEM10_020 and Eco/Bam 41 and 42 PCR products into pMLL4AC
Digests:

eco/bam: 41, 42

R: bgl/xhoI of igem019-gel purify (mapping at same time): 6181, 1607. Cut out top band.

L:bam/xhoI of igem001

digests started at 12:46pm

Gel purified 19b,d: There was a band at around 6000bp, but I couldn't see one at 1607, but that could be because I ran the gel for a long time. I gel purified the top band anyway and I'll continue on with the next step of assembly. I could map again, but I think my analytical colPCR combine with sequencing results will be a pretty good confirmation.

Zymo'd 41, 42, and 1.

Ligations:

41/42+pMLL4AC, transform into lefty 19b/d with 1, transform into jtk030

Started at 2:55pm.

Rescue started at 3:41pm.

PCR of ca998/g00101 off iGEM10_042 and 041 in hybrid vector
Analytical Mapping:

Lane 1: iGEM10_042, expected length: 2863bp Lane 2: iGEM10_041, expected length: 3093bp



The PCRs look successful. I'm going to Eco/Bam the PCR product and ligate with pMLL4AC.

Cotransformation Results for iGEM10_019 and ef1a/atub+2093
iGEM10_019: only sample c was co-tranformed, but I'm only going to miniprep b and d, since I forgot to streak sample a on the antibiotics plate.

All of the samples for ef1a/atub+2093 were co-transformed. I'll pick a lot more colonies today, colPCR, inoculate, and streak for cotransformation.

Amy N. Kristofferson 14:37, 22 July 2010 (EDT)
To do:
 * Plate at 4:35pm
 * Run colony PCR on gel
 * See if i have any ef1a MPs that mapped well that I could submit for sequencing. I want one w/o the double T deletion. However, it's prob before the atg used as a start codon, so it may not affect anything. We'll see...
 * On a second glance, mapping data for ig114/2271+2017 could be ok. Maybe do with iGEM10_043/044 (four reactions total) and submit them for sequencing?

Nuclear Localization check
pjc012 backbone

Bgl/XhoI transfer of CDS of our protein

his6tag prom-rbs-his6-my gene!

rfp+nls: 1884+1858 in 1601KC

Transformed into jtk049. Started rescue at 3:35pm. Plated on KC.

Tomorrow, pick colonies. Can do colPCR off plasmid (1601 series).

Next day: miniprep, map, sequence? PCR on bgl/xhoI sites.

may need to pcr a new bam/xhoI around GFP-Nls and then cut into mike's plasmid

Sequencing Results for iGEM10_047, 48, 51, 52 (SL/VB Assembly Product with L-Lysozyme Self Lysis
All parts look perfect for about the first and last 1000bp.

Picking ef1a/atub-Bjh2093 in pMLL/1601KC hybrid vector colonies

 * Pick four colonies of each.
 * ColPCR w/ ca998/g00101
 * Inoculate in LB-KC

Picking iGEM10_019 colonies
The plate looks great. It's covered in fairly healthy looking colonies.


 * Pick 4 colonies off of iGEM10_019 plate.
 * Run ColPCR w/ conor's oligos from Gibson:

If both 17 and 16 (a part of 18) are there, the colPCR will produce a band ~888bp long.


 * Inoculate in LB-AK

ColPCR results from above colony picks


Lane 1-4: iGEM10_019 w/ igemTen21/24, I wanted 888bp band, so they all look great! Lane 5-8: ef1a+2093 w/ ca998/g00101, expected 1952, all bad--last two are probably cotransformed w/ ef1a parent vector Lane 9-12: atub+2093 w/ ca998/g00101, expected 2190, all bad

Even though my ef1a/atub+2-93 colPCR results don't look so hot, I'll miniprep the ones that weren't co-transformed and map them tomorrow.



ColPCR results for ig114/2271+2017 picks
This analytical backbone PCR seems useless, since a 900bp band was supposed to reveal the presence of the p15 origin of rep, a sign that the part was in the hybrid vector but my negative control, a part in pMLL4AC, also produced a 900bp band! My only choice of analysis then is mapping, but all of the colony PCRs look very crazy. Maybe I'll just miniprep and map them all anyway. Another route I could do would be to perform an Eco/Bam transfer into a vector w/ diff antibiotics, and then do another Eco/Bam transfer into the PMLL4AC. That's just going take a long time...

th395/th396



ca998/g00101



Minipreped everything and started an analytical Eco/XhoI digest at 5:21pm.



One of these is clearly still in its hybrid vector, but I don't know what's going on with the single bands. I'm going to Fusion PCR off ___ using ca998 and g00101. Then I'll Eco/Bam that into PMLL4AC.

ig114/2271+2017 vector transfer redo
Pick 4 colonies for ig114/2271+2017. Normal colPCR seems to be a waste of time, but I'll try it anyway. I'm also going to do my backbone analysis PCR. Negative control: Bjh2345 in PMLLAC.

Assembly of iGEM10_018 and 017 to make iGEM10_019
Manual R/L Digest of iGEM10_018 and 017. Started at 10:00am. Zymo'd. Ligation started at 1:08pm. Transform into Righty cells. Rescue started at 1:50pm Plated on KA. Labelled plate iGEM10_019.

One Pot Assembly of atub/ef1a with jh2093
DNA used: Rigthy: Bjh2093 in 1601AC Lefty: ef1a PMLLKA b (7/14 miniprep) and atub PMLLKA b (7/14 miniprep) All samples are methylated.

NOTE: I'm intentionally creating a hybrid vector! I think this will save time. Just make sure not to transform into Righty, because then I won't be able to Eco/Bam the product into a good vector. Also, this ef1a has a two base pair deletion, so it may not be functional.

Digest started at 1:32pm. Heat kill at 2:49pm. Add ligase at 3:15pm. Add jtk030 cells and heat shock at 3:45pm. Start rescue. Plate at 4:49pm.

1uL lefty plasmid(methylated) 1uL righty plasmid (methylated) 1uL NEB2+ATP 0.3 uL XhoI 0.3 uL BglII 0.3 uL BamHI 6.1uL Water
 * Set up the digestion:
 * Digest for 1 hour at 37C
 * Heat kill at 65 for 20 min
 * Add 0.3uL T4 Ligase
 * Ligate at room temperature for 30 minutes
 * Put on ice for transformation
 * Transform, rescue, plate on dual antibiotic plates

Amy N. Kristofferson 13:45, 20 July 2010 (EDT)
None of the colonies picked yesterday from the iGEM10_047, 48, 51, 52, or 16+2294 were co-transformed. And since the ColPCR didn't reveal much of anything, I'm just going to miniprep the first two samples of each, except for iGEM10_047. iGEM10_047's d sample looked great on ColPCR, so I'll miniprep sample a and d instead of a and b. Also, somehow all 12 of the colonies that I picked for the transfer of 2271/ig114+ 2017 from the hybrid into PMLL4AC seemed to still be in the hybrid vector, based on my analytical pcr (they all had bands around 900bp). I'll miniprep four of each anyway and map them. If they're not the right side, I'll do the transfer over again.

To do:
 * Minipreps
 * Ask Tim about Ef1a sequence.

Picking colonies w/ Payload and Payload delivery
30µL into ea mL TB media (use TB for growing up for assay)

Miniprep Map


Submitted a sample a for each of the 2345/1968 assemblies for sequencing, except I submitted sample d for iGEM10_047.

16+2294 is righty methylated. That's why I only got one band. It's the right sized band though. I'll map it with Eco/XhoI. Started digest at 4:10pm.



Lane 1: 16+SLa Lane 2: 16+SLb

Expected lengths: 4644, 1466

Analysis: I think the top band is parent vector. Don't know why sample B has three bands... very strange. I submitted a for sequencing.

Redo of Hybrid vector transfer
Those oligos shouldn't PCR off the genome (http://archaea.ucsc.edu/cgi-bin/hgPcr?hgsid=462798&org=Escherichia+coli+K12&db=eschColi_K12&wp_target=genome&wp_f=cggttatccacagaatcaggg+&wp_r=gcgctgatgtccggcggtgc&Submit=submit&wp_size=8000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0)

I'm going to make more of pMLL4AC Eco/Bam digest from Tim's stock in plate 2, well F7 (pMLL4AC Bth8062). Started digestion at 2:04pm. Drop out is 813bp. Vector should be 2745bp.

Use 3uL of digested pMLL4AC instead of 1uL when ligating.

Unfortunately, there's only about 1.75uL of digest PMLL4AC vector left, so I used it to redo the ligation w/ eco/bam digested ig114+2017. I started the ligation at 1:51pm. Started rescue at 3:04pm.

Redo of ligation with new pre-digested and gel purified PMLL4-AC w/ FFGFP dropout. Started ligation at 4:45pm. Started rescue at 5:30pm.

Amy N. Kristofferson 14:51, 19 July 2010 (EDT)
Everything grew up very well. The only concern I have is that the colonies with 2345 in the part have a transparent loop around the colony, possibly suggesting that self-lysis is starting. I'll have to careful when I miniprep these.

I pick colonies, ran a normal colony pcr for all of them, ran an analytical pcr to make sure the parts I transfered out of the hybrid vector aren't still in the hybrid vector, inoculated and check for co-transformation for the assembly plates. Here are my notes:


 * pick four colonies off each plate.
 * run colPCRs w/ ca998/g00101 for all of them
 * run colPCRs w/ th395/th396 on ig114+2017 and 2271+2017 vector transfers and original minipreps to check for hybrid

Timing wise:
 * make colPCR MM for 5 rxns w/ th395/396.
 * set up tubes for colPCR (need 28 tubes for regular, and 8+2=10 for th395/396 version)
 * set up plate for co-transformation check: 5x4 grid
 * cross out gfp colonies on ig114/2271+2017 plates
 * set up two 24-well plates. Need 2AC-4AK-1AC LB.
 * pick colonies, colPCR (don't forget to double dip for the 2017s!), inoculate, co-transf. plate
 * don't forget to add template DNA from original minipreps for th395/6 PCR for comparison check.

Analytical PCR results for Transfer from hybrid vector
This is a gel from a colPCR with oligos th395/th396 of four colonies picked from 2271+2017 and ig114 7/16/10 plates. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. This gel suggests that all the colonies I picked contained the hybrid vector. Tim said that if I need to do the transfer over again, I should use more pMLL vector (~3uL). For now, I'll pick more colonies.



ColPCR
This results aren't very good. But I think that's to be expected when parts are about 6k or greater. I'm going to mostly ignore these results and miniprep a couple samples from each reaction anyway.

The analytical PCR makes it seem like all the hybrid transfers are still in the hybrid vector... :(





Amy N. Kristofferson 15:20, 16 July 2010 (EDT)
To do:

-Eco/Bam digest ig114+2017 g.p. and 2271+2017 g.p. Run on gel and gel purify. Ligate to pMLLAC pre-digested vector. (calculated expected lengths before running gel, to make sure it's worth the trouble. Bands might be so similar that maybe just a zymo is better)

-ligate g.p. 2294 with g.p. 16a.

-assemble SL-L with vacuole buster:
 * Bjh2345 in pMLL4-AC with iGEM10_043 and iGEM10_044 (I'm going to use my 2114+2275 miniprep from 7/13, even though it hasn't been sequenced yet. It mapped well, but this doesn't say much since Pcon is so small. Even so, I'm do the reaction anyway and trash it if the sequence comes back bad, like the last one did.)
 * Bjh1968 in pMLL4-AC with iGEM10_043 and iGEM10_044

NOTE: can't use one pot because iGEM10_043 and iGEM10_044 are NOT methylated. I transformed jtk030 cells to make them. I'll have to do separate righty and lefty digests.

Sequence Analysis of Ef1a/Atub
So where to go from here?

I'll submit atub for sequencing with g00101. As for ef1a, since my ef1a in the AK vector (was supposed to be KA... argh) sequenced perfectly. I'll just Eco/Bam Transfer it? Then I'll get colonies tomorrow, and I can ColPCR/inoculate and then miniprep on Tuesday. OR I could pick more colonies today. However, even if I map well, the part could have mutations...

Transfer of 2271/ig114+2017 to PMLLAC from pMLL/1601 hybrid
Eco/Bam digest of 2271+2017 g.p. a, ig114+2017 g.p. a.

Gel purify part:

2271+2017 in pMLL/1601 vector Eco/Bam: 3670, 2704 Lower band includes parts.

ig114+2017 in pMLL/1601 vector Eco/Bam: 3670, 2934 Lower band includes parts

When I pick colonies, I'll run two ColPCRs: ca998/g00101 and th395/th396. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. I'll also run the ColPCR on colonies that I know contain the hybrid so that I can compare. This will allow me to do less minipreps. I'll still map them though, to confirm the vector size.

TH395  gcataGAATTCcggttatccacagaatcaggg        fwd for EcoBAM p15A, pBth7011 TH396  ctaggGGATCCgcaccgccggacatcagcgc         rev for EcoBam p15A, pBth7011

Second Step of Assembly for SL/VB: L-1968/2345 to iGEM10_043/044
Bam/XhoI: 2345 in PMLL4 b, 1968 in PMLL4AC (T1, L) Bgl/XhoI: 2114+2316 g.p. a, 2114+2275 a (7/14)

Submit 2114+2275a 7/13 miniprep for sequencing
Submissions:

Results: Great! This part is as it should be.

Sequencing Results for ig114+2017
I'm going to Eco/Bam Transfer the part, because it's currently in a pMLL-jh1601 fusion backbone.

Amy N. Kristofferson 14:55, 15 July 2010 (EDT)
To do:
 * reinterpret mapping results w/ SL with 1601 plasmid
 * Design oligo for sequencing of 2271+2017
 * gel purify SL. Ligate to gp'd 16a
 * sequence analysis of ef1a and atub

Mapping Yesterday's Minipreps


Ran out the gel a bit more, and I think it shows that the two 2271+2017 samples mapped well:



Sequencing Results for the rest of the Minipreps from my first batch of colony picks from Gel-Purified Assembly
The boundaries of parts 2114+2275 and ig114+2017 look good. I'm going to PCR off the middle to check if the insides are go too. I used ca56 for ig114+2017 (binds to pbad promoter). I designed a primer for the middle of

Amy N. Kristofferson 13:24, 14 July 2010 (EDT)
To do:


 * Redo cotransformation of Tera'a Payloads with Payload delivery devices in 1601K. DON'T FORGET MG!!!
 * Run colony PCRs for KA transfer
 * Run colony PCRs for SL/VB Assembly colony picks
 * Miniprep everything that wasn't co-transformed

Sequencing Submissions: the rest of my gel-purified assembly minipreps
iGEM51-56= each part w/ ca998 and g00101

Cotransformation Redo
Rescue started at 11;32am.

Sequencing Results for 2114+2316 (iGEM10_043): Great news! Successfully put Pcon in front of VBa
I submitted the part with ca998 and g00101, and the results clearly show that the Pcon is in front of the vacuole buster. Woo hoo!

KA transfer
Christoph Miniprepped everything he needed.

I miniprepped the remaining 1mL of 10 or bjh2294-PMLLKA (eluted with 25uL to keep normal MP concentration), ef1a-PMLLKA b, atub-PMLLKA b,c.

Parent Vector bleed-through results: All samples from 1,2,3,4,6,7,8,12,13,16 are good. The only samples with co-transformation were 15a and 9a,b,d.



Colony PCR- ca998+g00101
NOTE: lanes 49-96 refer to the middle gel. Just subtract 48 from the lanes in the table to see what number to refer to on the actual gel.

Colony PCR-th313+iGEM10
Note: the results for 5 are in the middle right gel shown above.



SL/VB Assembly Colony Picks
Cotransformation results: 2h and 6a were co-transformed. Everything else had no growth on the triple antibiotics plate.





Co-transform jh2348/2291-1601K with Tera's Payload
Added 100uL of H2O to 1uL of DNA from Tera's Ef1a and Atub payload constructs. Added 10uL of H2O to 1uL of DNA from my minipreps of 2348/2291-1601K.

Added 1uL of each of those dilutions to 70uL of MG1655 cells.

Mg needs to be added for all steps of cloning the PDD's 1M MgSO4 10µL into rescue step (after heatshock) 400µL spread on plate (if +Spec, add 20µL and spread) 30µL into ea mL TB media (use TB for growing up for assay)

Started rescue at 4:06pm.

KA Vector transfer: ColPCR (parts and vector backbone check), inoculate, grow up
I picked colonies, inoculated and ran a colony pcr for all the parts we moved into the new, CORRECT KA vector.

Important notes:
 * 5 and 11 were originally in AK vectors. 10 was also in an AK vector, but I gel purified it. Even so, I will run an analytical PCR of my minipreps of 10, and I ran an analytical Colony PCR with th313 and g00101 for my 5 and 11 colonies. I picked 12 colonies of 5 and 11.
 * I also check for parent vector bleed-through by plating all the parts on spec, except for 9 (plated on triple antibiotics since it came from CK) and efla+atub (PCR products)
 * All the inoculations are in the shaker.
 * The colony PCRs are in the black PCR machines. They'll probably be done around 6pm.

SL/VB Assembly: Picking colonies from 7/6, 7/7, and 7/8 plates
Picked 8 colonies from Assembly attempt #2 (one pot) and assembly attempt #3 (gel purification) for 2114+2275, ig114+2017 and 2271+2017. Inoculated, checked for co-transformation, and set up ColPCR.

IMPORTANT UPDATE: TUBE MARKED PMLL6 (KA) in Eco/Bam Box is INCORRECT
Christoph just ran and analytical digest/gel that proved that the vector in the tube that was supposed to contain KA actually contained AK vector instead of KA. I need to Eco/Bam transfer the parts into a new KA vector. Also, this explains why the assembly with the Self-Lysis Device didn't work. We need to Eco/Bam transfer it into a KA vector too.

Digesting the new pMLL-KA backbone
Marianna gave us 10uL of her CMED7 in KA part. The dropout will be 324bp. To digest all of it, I multiplied the Analytical Mapping protocol by 2.5:

10uL of Water 10uL of CMED7-PMLLKA Miniprep 2.5uL NEB 1.25uL EcoRI 1.25uL of BamHI Total: 25uL Load 12.5uL into 2 wells. Gel Purify. Elute each with 12.5uL of H20.

Started digest at 4:30pm.

Ligation of ef1a and atub (bjh2342) eco/bam zymo'd digest
Ligated ef1a and atub eco/bam zymo'd digests (from my previous insertion into the supposed KA vector) with the new gel purified Eco/Bam digested KA vector. Started at 7:05pm. Transformed jtk030, MC1061 pir+ cells. In retrospect, I could have used Lefty.

Checking vector backbones of SL/VB Eco/Bam transfers
Set up a 2K55 PCR reaction with Taq MM, 1uL of 1/10 th313 oligo (in the middle of AmpR) and iGEM10 (rev oligo right before EcoR1 site) to verify the order on antibiotics on the backbone. This reaction PCR's off the AmpR gene to right before the EcoRI site. So if the vector is actually A-, the PCR product will contain a bit of AmpR and plasmid backbone and will be 865bp. If the plasmid is -A, the PCR product will contain a bit of AmpR, lots of plasmid backbone and the other antibiotic. In the case of CA, this amounts to 1808bp.

See Eco/Bam page in Excel notebook.

This data suggest that all the backbones that should be A- are indeed A- and that all the backbones that are -A are indeed -A. So the antibiotics shouldn't be the problem. For whatever reason, nothing turned up in the 2345 lane. I'll run the PCR again just to double check.





Lane	Part	Color	Expected length	Good? 1	Bjh2345a 	AC	865	no-empty lane 2	Bjh2345b	AC	865	good 3	Bjh2345c	AC	865	good

Threw out Bjh2345a. Must be a bad miniprep. I should probably map b and c. I guess I haven't yet...

Analytical digests of SL/VB Gel Purf. Assembly Minipreps
Eco/XhoI

Eco/BamHI

Payload Delivery: Analytical Mapping of 2348 and 2291 in 1601K
Both parts look great! I'm going to give them to Jin.



Amy N. Kristofferson 21:13, 10 July 2010 (EDT)
Must do today:
 * Miniprep good colony pcr/cotransformations from yesterday
 * Pick colonies for Tahoura

If I have downtime, could start items in the above To Do list

From yesterday's colony picks, only the first colony picked from 16a H3 was cotransformed. No idea how that happened, since I gel purified the digests before ligating.

Colony PCR of SL/VB and 16a+SL Assembly
Results:

Very odd. Even though I gel purified the correct bands, it appears that only 2114+2316 worked correctly. I'm going to check to make sure that everything is in the correct vector, because I don't know what else could be causing a problem. Since I'm miniprepping a the couple correct products, might as well miniprep and map a few others to try and figure out what the problem may be.



Animal Promoter: Analytical Mapping of atub and ef1a promoter in pMLLKA
Started an Eco/Bam digest of the 4 minipreps I made of each promoter (all of which had GREAT colony PCRs) at 10:35am. They will be ready to run on a gel at 11:05am.



Once it has been verified that these parts map well, I'll submit them for sequencing and then begin the next step of assembly.

Submitted ef1a-a and atub-b

Sequencing submission:

Cotransformation check
None of the 2271+2017 or ig114+2017 colonies were cotransformed, but ALL of the iGEM10_018 (16a+SL) were cotransformed.

Colony PCR results for 2271+2017, ig114+2017 atub and ef1a promoter, iGEM10_16a
Can disregard the results for 16a+SL since all samples were cotransformed. 2017 combo inoculations are useless since nothing showed up on the colony pcr. Very strange, since they weren't co-transformed. GOOD NEWS: atub and ef1a look great.



Redo of SL/VB and 16a+SL digests with Gel Purification
To avoid co-tranformation and hopefully encourage the creation of the parts I want, I'm going to run a preparative gel, select the appropriate bands and gel purify.

Performed a Lefty (Bam/XhoI digest), Righty (Bgl/XhoI digest), and one-pot digest on all the parts in my SL/VB promoter as well as iGEM10_016a and Bjh2294 in PMLL-KA can be found in the Cheshire Chat plate in H3 and F3. I'll gel purify the Lefty digests of Bjh2345, Bjh2271, Bjh1968, Bjh2114, ig114, 16a and the Righty digests of Bjh2316, Bjh2275, and the two self-lysis devices. The other digests are just to make sure everything was methylated correctly and that the one-pot reaction works on these vectors.

Notes on protocols: Used Manual 2ab Assembly protocols for Lefty and Righty Mastermixes. Used my One-pot recipe, except with 0.4uL of each enzyme. Also, the DNA taken from the Cheshire Cat Plate was diluted by half b/c it was eluted with 100uL, so we had to adjust the amounts of NEB2 and enzyme used so that the recipe in the R/L digests turned out to be 1.6uL NEB2 and 1.5uL of each enzyme total, since our using 8uL of DNA instead of 4uL of DNA would otherwise have diluted the buffer and enzyme.

Digests started at 3:35pm.

From left to right: Lefty Digests, Righty Digests



Triple Digests



Results:

NOTE: lane 2 in the lefty gel is Bjh2294 H3, and everything else is pushed over one lane

Summary: Everything is methylated as it should be. I cut out all the right bands and purified them. Also, this shows that triple digestion isn't very efficient and cause a lot of parent vector bleedthrough (the top band of all the lanes). Also, note how dilute the SL DNA is... I'll still elute with the standard 8.5uL to concentrate the DNA, but this is still a potential problem.

Ligation and Transformation of SL/VB and 16a+SL purified digests
Started ligation at 6:45pm. Will transform into jtk030 (pir +, MC1061 cells). I forgot to digest 2017, so I used a righty digest that I had done previously. Started rescue at 8:10pm.

Amy N. Kristofferson 13:45, 7 July 2010 (EDT)
All the samples from 16a,b + SL were cotransformed, and since Jin mentioned that making a mastermix for the onepot reaction may result in there not being enough enzyme per reaction, I'm going to do it over again without making a mastermix AND using 0.5uL of enzyme instead of o.3uL of enzyme. This should cut back on parent vector bleedthrough as well.

There are a significant number of colonies on the plate for the 2271+2017 and ig114+2017 redo. I'll pick colonies/colony pcr/inoculate/check for cotransformation for 8 colonies from each. Also, since all the colony PCR's failed for my re-picking of 2114+2275, I'm going to redo the one-pot reaction for that as well.

The ligation of the atub promoter to the KA vector (Bjh2342-pMLL6-KA) was very successful, since I got a lot of colonies. I'll inoculate and run colony PCR (no need to check for co-transformation. Since my two colony picks of ef1a seemed to fail on the colony PCR yesterday, I'm going to pick a bunch more, inoculate, and run colony PCR again.

SL/VB Assembly: Redo of One Pot (with more enzyme) for 2114 combos

 * One Pot (used 0.4uL of enzyme):
 * 2114+2275
 * 2114+2316
 * Will be ready for ligase at 2:00pm. Added ligase at 2:00pm. Will be ready for transformation into RIGHTY cells at 2:30pm. Rescue started at 3:30pm.

iGEM10_020 Assembly: redo of 16a+SL assembly, starting with new L/R digests

 * Redo of L/R digest of 16a and 6+24
 * Put in incubator at 12:14pm. Ready for Zymo at 1:14pm. Zymo'd and added ligase at 2:00pm. Will be ready for transformation into RIGHTY cells at 2:30pm. Rescue started at 3:30pm.

Possibly successful assemblies (Animal Promoter, SL/VB and iGEM20

 * Picked colonies (8 each)/inoculate/cotransformation check
 * 2271+2017
 * ig114+2017
 * 16a+SL (pick at least 12 NEW colonies)
 * atub and ef1a (no cotransformation check)

Miniprep of Payload Delivery Vectors
Miniprepped Payload Delivery stuff (in shaker w/ Mg LB)

Sequencing Results for iGEM10_017b, 2114+2316b,Bjh2345 PMLL4
Sequencing results:
 * iGEM10_017b: the first 990bp or so of the 1546bp part are perfect, so I think it's safe to say that this miniprep is good to use for future assembly.
 * 2114+2316b: this proved to have the Pcon, but no vacuole buster in the part. At first I thought this was odd, since the part miniprepped well, but it makes sense now. The part had a bad colony PCR since only the 43bp pCon was in the vector. The part had what looked like a good miniprep map since the vector was the same size as the part, and I when I saw a band, I assumed it was the part AND the vector. My mistake.
 * Bjh2345 PMLL4: bad read. Do again, but make sure concentration is accurate?

Colony PCR results for all colonies picked today
Note: they ALL look horrible. Bands in the 16a lanes are probably from cotransformation with 16a vector, since that part is about 850bp.



Transposase Assembly: Colony PCR of iGEM10_016a,b with Self Lysis Device
Picked 4 colonies of each. Ran Colony PCR. Inoculated. Plated to check for co-transformation.

Animal Promoter: Colony PCR of Ef1a
Picked 2 colonies. Ran Colony PCR. Inoculated.

Conor Eco/Bam digested and zymo'd the atub promoter (Bjh2842). Started ligation with PMLL6-KA vector at 4:45pm. Transformed into jtk049 cells. Started rescue at 5:55pm.

Payload Delivery
Since the payload delivery device contains the Self Lysis device under a promoter that's induced in low levels of Mg, I need to grow up my colonies in LB containing 30mM Mg to prevent Lysis. In the future, I should also rescue/plate on Mg. I selected clear colonies.

Tomorrow, I will miniprep the inoculations and map the minipreps next to the parent vector containing the part, so I can make sure the whole part is in the new vector. (Jin said recombination events can occur which could shorten the part)

Redo one pot reaction with 2271+2071 and ig114+2071
Conor set up two one pot reactions and put them in the thermocycler under my ONEPOT program. Added ligase at 4:45pm. Will transform into LEFTY cells. Started rescue at 5:55pm.

Picked more colonies for 2114+2275, since miniprep mapping failed
Pick 8 more colonies from 2114+2275. Ran colony PCR, inoculate, check for cotransformation.

Summary of Results so far
For whatever reason, my Colony PCR results and miniprep results don't seem to ever correspond.

SL/VB Promoter Assembly: Mapping the minipreps produced from Round 1 and 2345 Redo
Started digestion at 5:18pm. Will be ready for the gel at 5:48pm.



Transposase Assembly: Ligation of iGEM10_016a,b Lefty digest with 6+24 Righty digest
Started ligation at 4:05pm. Ligated unmethylated 6+24 digested with BglII+XhoI with both unmethylated iGEM10_016a+b, which had been digested with BamHI+XhoI. Tubes are labelled 16a+SL and 16b+SL (SL meaning Self Lysis, which is part 6).

After ligation is over, I will transform into RIGHTY cells. Started rescue at 5:05pm. Plated on CA.

Animal Promoter: Ligate Ef1a Eco/Bam digest of PCR product with PMLL6-KA
Started ligation at 4:05pm. Ligated Ef1a Eco/Bam digest of PCR product with pre-digested PMLL6-KA.

After ligation is over, I will transform into LEFTY cells. Started rescue at 5:05pm. Plated on KA.

Payload: Ligation of Bjh2348 and 2291 with Pjh1601K
Started ligation at 4:05pm. Ligated Eco/Bam digests of Bjh2291 and 2348 with my Eco/Bam digested Pjh1601K plasmid.

After ligation is over, I will transform into jtk049 cells, because they don't need to be methylated. Started rescue at 5:05pm. Plated on K.

Transposase Assay Take 2: Testing time for Transposase expression and time for transposase activity

 * 1) Each inoculation contains about 4mL of cells. Move 1000uL of cells into four flasks.
 * 2) Each flask contains 900uL of cells.
 * 3) Add 10uL of arabinose for each mL of cells at t=o, 1 hour, 2 hours, 3 hours.
 * 4) Since arabinose is 100x, will add 9uL of arabinose to each flask
 * 5) Added first 9uL at 10:49am.
 * 6) Added second 9uL at 11:49am
 * 7) After four hours have passed since you first added arabinose, add 1uL of atc per mL of cells. Allow 1 hour for lysis.
 * 8) Will add 0.9uL of atc, since 900uL of cells are being used and the concentration of the atc is 1000x.
 * 9) After that one hour, add 5uL of DNA to each tube.
 * 10) Remove a 200uL sample of lysate after 30min, 1 hour, 1.5hour, 2hours.
 * 11) Immediately after removal, spin down the lysate and zymo the supernatant.
 * 12) Transform lefty pir+ cells with the lysate. Plate on Amp and Gen.

Unfortunate update: Sequencing proved that iGEM10_020b is a dud. Gibson didn't work because one of our junctions contained palidromic terminal repeats, so the oligos designed based off those junctions didn't have a unique binding site. Oh well. We'll proceed with manual 2ab assembly.

Mapping of iGEM10_016 minipreps a and b
An Eco/Bam digest of iGEM10_16 in PMLL5-CK should result in bands at 2662 and 867.

Note: iGEM10_017 has already been mapping. (see below) Results were slightly confusing, so I'll submit it for sequencing...? iGEM10_001 was also mapped, and looks good. 1a-c are all good options.

Started digestion at 4:55pm.



Both have bands at the appropriate places, but 16b has a lot more parent vector remaining, for whatever reason, so 16a is probably the best choice.

Assembly of iGEM10_018
Lefty Digest of iGEM10_16

Righty Digest of Self Lysis Device (6+24 taken from well G10 of ROMEO plate) Started at 4:42pm. Zymo'd and stored in my working box.

SL/VB Promoter Manual Assembly: Analysis of One-Pot reaction
2114+2316 and 2114+2275 both successfully transformed righty pir+ cells. The plates are covered in colonies. 2271+2017 and ig114+2071 weren't as successful. The former had no colonies, while the latter had 11, which is surprising, because I used barely any DNA.

I will pick four colonies from each, colony pcr, inoculate, and check for cotransformation.



Cotransformation results:



The ones with letters in the corners were chosen for minipreps.

Moving into jh1601K
Performed an Eco/Bam Digest of jh1601K. Put in incubator at 11:09am. Trashed that digest. Realized I need to do a gel purification, so I'll do the digest again with 4uL of plasmid.

Started digest again at 4:54pm.

2345 Redo from 24+PMLL4 digests
The plate is covered in colonies. I will pick 8 colonies, inoculate, and check for cotransformation.

Eco/Bam transfer 2348 and 2291 into 1601K
Started Digest at 12:19pm.

Zymo'd and stored in my working box.

2345 Redo: Ligating Tim's ebid 24 and EB pMLL4-AC
Since my Colony PCR turned out so horribly yesterday, and 2345 is the Self Lysis Device we've been using for all our construction, I'm just going to use Tim's digests and ligate them together.

Started ligation at 11:48am.

I transformed the plasmid into lefty pir+ cells. Started rescue around 12:30pm. Plated on AC at 2:13pm.

One Pot Assembly of Jin's Parts
I'm going to redo the construction of the following with a one pot reaction, since they're all methylated.

Started digest at 11:10am, for all but ig114+2017. I added the speck of 2017 (~.2uL) left at around 11:36am. Started the 20 min heat kill for all but ig114+2017 at 12:30pm. Accidently set the heat kill program for 20 sec instead of minutes and added ligase. Hopefully that won't hurt anything. Put all four tubes in the PCR machine to heat kill at 12:53pm. Added ligase at 1:41pm. Started rescue at 2:42pm.

These numbers have been based on Qiagen minipreps of Jin's pBjh1601 assembly vectors: 1uL lefty plasmid(methylated) 1uL righty plasmid (methylated) 1uL NEB2+ATP 0.3 uL XhoI 0.3 uL BglII 0.3 uL BamHI 6.1uL Water
 * Set up the digestion:


 * Digest for 1 hour at 37C
 * Heat kill at 65 for 20 min
 * Add 0.3uL T4 Ligase
 * Ligate at room temperature for 30 minutes
 * Put on ice for transformation
 * Transform, rescue, plate on dual antibiotic plates

Notes: tubes labeled with a red slash distribute the plasmids into it 1hr at 37 and heat kill steps of the procedure
 * This requires the use of BglII and BamHI methylating strains!
 * 10X "NEB2+ATP" is a homemade buffer present as aliquots in 500uL
 * You can make up a mastermix of the enzymes/buffer/water and
 * The leftmost thermocyler in 327 has a program JCA/1123 that runs the

Eco/Bam digest of EF1a PCR product
Started digest at 11:55am. Zymo'd the product.

I need to figure out what assembly vector to put it in and then map and sequence the part to make sure it's correct.