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Team:NYU/Notebook
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Notebook
7/20/10
- John:
Performed digestion of 32 plasmids using either EcoRI, PstI, or both. These plasmids included RBS, Flag, E1, F1, sectag (multiple), NUB, and Strep. This was performed to see if our digestion enzymes were working correctly. After digestion, these solutions were run in 4 1.5 gels with large sized wells to verify if the enzymes were cutting.
7/21/10
- John:
Ran 1.5 % agarose gel to verify whether primers worked on the Nub-Fusion construct. Previously, we PCRed NUB-Fusion plasmid using verification primers. After, we used this product as a template for PCR with the NUB specific primers that we designed. Sec-tag was also PCRed and run on the gel. Additionally, old PCR cleanup NUB and Sectag were run on the gel to see if they can be used or disposed.
- Harris:
Transformations from the day before of ADH1-Aga2Linker and ECFP-Terminator in the Biobrick Chloramphenicol plasmid failed, although the retransformation of Aga2 (in the Amp plasmid) alongside them worked. Ligations left overnight so today I am retransforming them alongside BBa_K098994, which is 5B from Plate 3 of the initial distribution, and is in a Chloramphenicol resistance plasmid. This should grow up fine, and if it does, then we know that the problem is with the ligations, but if it doesn’t, then there is a problem with the Chloramphenicol plates.
7/22/10
- Russell:
PCR results from 7/20 were disappointing but not unforeseen. Previous sec tag PCRs show band of appropriate size, but biobricking attempts have been unsuccessful with cause unknown. Will perform one more biobricking procedure on the sec tag under careful scrutiny before troubleshooting for larger problems.
Transformed the N-terminal and C-terminal GFP biobricks from the initial distribution. These will be used to show that the test scFv and test antigen are, in fact, complexing in the cytoplasm (and maybe on the surface).
Miniprepped the Gal4 and VP16 biobricks to use as a Gal1-10 promoter activator for the response system.
7/25/10
- Russell:
Harris’ transformations of pADH1:Aga2-Linker and ECFP:Terminator assembly were successful. I inoculated 4 colonies from each assembly product in LB+Chloramphenicol and performed colony PCRs.
Unfortunately I REALIZED THERE IS NO RBS BETWEEN THE pADH1 PROMOTER AND AGA2! This simple mistake prevented the MIT team last year from having a successful system so we need to watch out for this. We may want to ligate an RBS onto each of the promoters we want to use and use each of those instead so we definitely do not make this mistake.
Retransformation of the secretion tag biobricking ligation was also successful. I inoculated 6 colonies from the two plates and performed colony PCR.
Transformation of the split-GFP biobricks (N-terminal and C-terminal halves of GFP) from the iGEM initial distribution was successful – inoculated 3 colonies of each biobrick but had already started thermal cycler, so colony PCR will be done later.
Ordered second set of Nub primers as well as biobrick-compatible primers for manipulating the backbone of pCTCON.
7/26/10
- Russell:
Got sequencing results from Genewiz for both of the supposed N-ub fusions we received from iGEM – neither sequence contained Nub, apparently both plasmids are void.
Instead of excising from a plasmid, we get to build the part via overlapping PCR (which, luckily, I’ve been wanting to try for awhile). So I broke the ~140bp Nub gene down into 3 overlapping sequences and ordered the appropriate oligos for the PCR.
7/28/10
- Russell:
Received both orders from IDT. Performed two overlap PCRs with different concentrations of oligos. Will run on a gel tomorrow with some Biobrick verification PCRs.
- John:
Research potential providers of scFv and antigen parts. Made transformation media and SOB.
7/29/10
- Russell:
Constructed the pCTCON plasmid backbone with BIOBRICK ends through PCR amplification. [extremely long cycle - 5:30 ext. time]. Also verified biobrick Aga2:Linker:LoxP via VerF/R2 primers and Aga2F/VerR2.
- John:
Email out requests for potential scFv and antigen parts.
7/30/10
- Russell:
I am trying to develop a couple of new (at least for me) protocols to use with biobricks.
The first is overlap PCR of the N-ubiquitin biobrick. This biobrick is only 140 bases long, so it is not viable to synthesize it via GeneArt (even though we still have all our basebucks) because of the much larger minimum order requirement. Instead, I broke the part down into three overlapping oligos and ordered them from IDT. The first oligo is just the first 60bp of the CDS. The second and third are the next 60bps (with 10bp overlap) but they are taken from the reverse complement of the sequence. I am currently writing a perl script to do this automatically and will post it as soon as I have it.
The second protocol I’m developing will help with maintaining stocks of biobricks. We are in full swing at the moment and keeping our stocks of soluble biobrick DNA is troublesome. Instead of using a good amount of each of our biobrick minipreps in an assembly, it may be possible to use a PCR product of the VerF/R2 primers and a biobrick template. That way we can just amplify the biobrick region from biobrick template and use the PCR product (with or without purification) directly in our assembly digestions and ligations. I just started the PCR cycle and will post results when I have them.
8/02/10
- John:
Emailed Dr. Marasco about anti-Tat ScFv, potential for this ScFv to work in the reducing environment of the cytoplasm. Also, researched potential sources for other “intrabodies,” stumbling on “Single Domain intracellular Antibodies: A Minimal Fragment for Direct In Vivo Selction of Antigen-specific Intrabodies”: Further thought required on the applicability of intracellular single variable domain (IDab).
- Russell:
N-ubiquitin biobricking transformation resulted in only one colony on the plate with 200uL of transformant. I inoculated it, but such a low efficiency may mean that it is a self-ligated plasmid (although self-ligation control resulted in no colonies).
pTEF:RBS assemblies from PCR amplicons resulted in many colonies. I inoculated 4 colonies and performed VerF/R PCR test to see if inserts are the expected size. Alongside this PCR I am making another 50uL stock of pCTCON backbone, so the cycle will be overnight and will run the gel tomorrow.
Literature research has turned a new leaf. Research into intrabody scFv has yielded a lot of great information and previous studies similar to the functional aspect of our system. Previous studies, however, do not use the split ubiquitin system nor encourage transcription of nutritional markers for directed evolution toward antibody binding (rather, they merely mutate via error-prone PCR and scan for binding antibodies through a system more like the two-hybrid). John is writing to researchers requesting scFv intrabodies and antigens, as well as a library of scFvs mutagenized from the original intrabody scaffold. Hopefully these requests will result in useful materials and advice!
- Harris:
Miniprepped pTEF-RBS from ligation of PCR product digested pieces, along with minipreps of the FLAG tag grown up and transformed from the initial distribution.
