SBB09Ntbk-Madhvi Venkatesh

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Madhvi Venkatesh 14:54, 2 February 2009 (EST)

Today, I got my project ball and I am making a new flagellar display system. I need to consider several possibilities and figure out what would be best to make and the source. The project description if on this page: new flagellar display system

Madhvi Venkatesh 15:40, 9 February 2009 (EST)

Possible places to find EtpA homolog (from CTEC):

APEC 01:k1
EHEC 0157:H7
Shigella flexneri

Madhvi Venkatesh 15:24, 23 February 2009 (EST)

So I rewrote my construction files after I realized that I can't display a protein on the N and C terminal ends of fliD because they are not facing the surface. The new versions of the construction files including Chris's notes on them are linked from the construction files page. We got oligos last Wednesday and I set up PCRs for the N and C terminal parts and wobble PCRs for the linkers. I purified and digested over the weekend. I also set up the SOEing PCR at the end of last week. Today I ligated the parts into pBca1256 and transformed. I will pick colonies tomorrow and send for sequencing on Wednesday. I also purified and digested the SOEing PCr for removing the restriction site in the N terminal fliD part.

Here are gel pics from the PCRs:

Here is the result of the SOEing PCR for the M1000 part {<N.fliD!} with the restriction site removed:

MV 2-23-09-1.jpg

Here are the results of the other PCRs:

MV 2-21-09.jpg

Madhvi Venkatesh 21:23, 27 February 2009 (EST)

I digestion mapped my basic parts:

MV 2-27-09.jpg

Lane Plasmid Clone Enzyme 1 Enzyme 2 Expected Bands Result
1 pBca1256-M10000 1 EcoRI XhoI 986 + 2113 correct
2 pBca1256-M10000 2 EcoRI XhoI 986 + 2113 correct
3 pBca1256-M10001 2 EcoRI XhoI 986 + 2248 correct (not completely digested)
4 pBca1256-M10002 2 EcoRI XhoI 986 + 1585 No DNA
5 pBca1256-M10002 1B EcoRI XhoI 986 + 1585 correct (not completely digested)
6 pBca1256-M10003 1B EcoRI XhoI 986 + 1582 correct (mostly undigested)
7 pBca1256-M10003 2B EcoRI XhoI 986 + 1582 correct

Based on this, I am sending M10000-1, M10001-2, M10002-1B, M10003-2B for sequencing.

Madhvi Venkatesh 14:46, 2 March 2009 (EST)

I got my sequencing data back and the results are up on my anderson lab sequencing page. M10000 and M10001 sequenced correctly and these clones are good to use. The wobble products, M10002 and M10003 did not sequence so well. The read for M10002 had Ptet, BBb scar and another short sequence in between the BglII and BamHI sites. The read for M10003 didn't have BglII or BamHI sites and just went from the EcpRI site to the part of the vector after the BamHI site. I double checked the protocols and it looks like I did everything correctly, so I think that it failed at the PCR step. I talked to Chris about the sequencing results and the low colony yield, and he suggested that I should try it again with Taq and/or DMSO and drop the annealing temp to 45. I set up four sets of reactions (one of each type for each wobble reaction):

  • Taq/55/no DMSO
  • Taq/55/DMSO
  • Taq/45/no DMSO
  • Taq/45/DMSO

The recipe I used for the reactions with DMSO:

 24 uL water
 5 uL Taq Buffer
 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc)
 5 uL Oligo 1 (100uM)
 5 uL Oligo 2 (100uM)
 0.75 uL Taq DNA Polymerase

Madhvi Venkatesh 03:19, 12 March 2009 (EDT)

So I got virtually no colonies on the plates from all of those conditions. Chris was suspicious and told me to thoroughly check the construction files and the sequences on the sides of the tubes. Everything looked right, so he told me to go ahead and redesign the experiment using SOEing instead of assembly. I wrote up the new construction files on my construction file page and ordered the new oligos. I set up the first round of PCRs today and ran them on the following gel. Fragments A,B,C,and D are in the first four lanes in that order:

MV 3-11-09.jpg

I think all of the fragments are the correct size (even though the ladder is a bit wierd) and I am going to set up the second set of PCRs right now with these fragments.

Madhvi Venkatesh 03:17, 22 March 2009 (EDT)

So, it turns out that those were not the correct sized fragments (the bottom part of the ladder is not visible in the picture above). When I ran the second set of PCRs, I got no product because the fragments that I had purified were the wrong things. I tried again with and without DMSO at 45 and 55 and at 55 with Taq and Phusion, but all of the times, my gel pretty much looked like this:

MV fliD PCRs.jpg

Chris told me that PCR with things longer than 60bp (which is these oligos) sometimes comes out wierd. I gues maybe there is some wierd secondary structure thing with these linkers.

I also ran a gel with a few of the wobble PCR products that I had from before that I had tried with Taq with and without DMSO at 55. I ran 5uL of each on a gel and the PCR appears to have worked; here's the gel (the order is mv47 w/o DMSO, mv47 w/DMSO, mv48 w/o DMSO, and mv48 w/DMSO):

MV mv47 and mv48 PCRs.jpg

I digested/ligated and tranformed mv47 w/o DMSO and mv48 w/DMSO, but again saw just a couple of colonies for each. It doesn't seem to be working for some reason and I can't figure out why. Maybe what I saw on the gel were primer dimers or something, but I feel like those wouldn't be that bright.

Anyway, the plan of action from here is to find a few new linkers over spring break, design the appropriate oligos and then do the construction when I get back. New construction files, etc. will follow.