Sambu/notebook
- Brainstorming:
- I have for sometime thought of producing synthetic spider silk proteins that would be modified to have RGD (Arg-Gly-Asp) residues to facilitate mammalian cell attachement. Traditionally, biomaterials scientists incorporate these residues using chemical reactions prior to cell seeding. If we were to encode these modified silk proteins in a DNA sequence optimized forprotein ,achinery in E coli, we would be well on our way to exacting the kind of control we need to modify these materials for various medical applications.
- So, thus far, I have made one such monomer with adequate provision for RGD repeats. We could use the E coli quorum sense to harness the E coli machinery for production of such proteins. From the quorum-sensing group's results, it appears that the point of optimum signalling and reception may very well coincide with the point n time when the cell's protein machinery is at a max -- so why not use that to our advantage and produce proteins of interest when and if the cells are at close vicinity to each other and are primed for protein-o-genesis.
Stuff done thus far:
- Did this experiment to verify that OD and cell dilution factors were linearly related>
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- Performed assays for the expression of his and strep tags on the E.coli surface. Used denaturation electrophoresis for this a picture is shown below:
protein denaturation electrophoresis gels
Description: 6/27 denaturation electrophoresis gel
The rightmost lane is the ladder
lane 1 Ladder
lane 2 OmpA1 + strep before induction with IPTG
lane 3 OmpA1 + his6 before induction
lane 4 OmpA1 + strep after induction
lane 5 OmpA1 + his6 after induction
lane 6 OmpA1 + his6 after induction
lane 7 OmpA1 + strep after induction
lane 8 OmpA1 + his6 before induction
lane 9 OmpA1 + strep before induction
Note that there were 2 copies of each sample: samples in lanes 2 to 5 were denatured for a 2 minute period as per protocol while samples in lanes 6 to 9 were denatured for a 10 minute period, Sammy S.
2. Then went on to investigate the appropriate times to induce expression of the OmpA-fusion protein constructs on the E.coli surface using IPTG
Week 3 gel
Gel 2: induced immediately
Lane 1: Ladder
Lane 2: Strep No IPTG
Lane 3: Strep denatured after 30 min
Lane 4: Strep denatured after 60 min
Lane 5: Strep denatured after 90 min
Lane 6: Strep denatured after 120 min
Lane 7: His No IPTG
Lane 8: His denatured after 30 min
Lane 9: His denatured after 60 min
Lane 10: His denatured after 90 min
This result seems to indicate that immediate induction and denaturation (stoppage) at 30 mins gives maximum expression of proteins. But we suspected this to be an artifact so we redid the assay again with a higher concentration for the samples
Gel 1
Lane 1: Ladder
Lane 2: His 0 min induction
Lane 3: His 30 min induction
Lane 4: His 60 min induction
Lane 5: His 90 min induction
Lane 6: His 120 min induction
Lane 7: Strep 0 min induction
Lane 8: Strep 30 min induction
Lane 9: Strep 60 min induction
Lane 10: Strep 90 min induction
Lane 11: Strep 120 min induction
Lane 12: His 0 min induction 120 min denature
Gel 2 samples run again
Lane 1: Ladder
Lane 2: Strep induced at 0 min
Lane 3: Strep denatured after 30 min
Lane 4: Strep denatured after 60 min
Lane 5: Strep denatured after 90 min
Lane 6: Strep denatured after 120 min
Lane 7: His induced at 0 min
Lane 8: His denatured after 30 min
Lane 9: His denatured after 60 min
Lane 10: His denatured after 90 min
Lane 11: His denatured after 120 min
Lane 12: His denatured after 120 min
LIGATING RANDOMERS ONTO THE PART OF OMPA'S LOOP 1
Started off with PCRs to pcr-out OmpA from 2 templates: +K-12 genomic DNA +OmpA in Topo plasmid (Kan/Amp resistant) For the first few days, this procedure gave negative results. Then we changed the protocol a little bit:
- Denatured for 2 mins as opposed to initial 10 mins
- Used fresh dilutions of primers (final conc. was 10 uM)
- Extension temp lowered to 68 degC as opposed to initial 72 degC
- These changes gave us good results as seen in image below:
The rightmost lane is the ladder
lane 1 Ladder
lane 2 BB OmpA from K-12 genome
lane 3 BB OmpA from Topo plasmid
lane 4 BB OmpA from K-12 genome
lane 5 BB OmpA from Topo plasmid
NB that all the pcr products here have BB ends
- The next step will be to use overlap pcr to create a pcr product that will have the randomer inserted at the beginning of OmpA. So we've ordered for a forward primer with the random (15 mer) library on it. This will be an ingredient in the 2nd step where we use it together with a reverse primer that takes care of the very end of OmpA.
- The first step will therefore utilize the forward primer matching the beginning of OmpA and the reverse primer going only until just before the beginning of OmpA.
- The two pcr products will be ligated with, say, SpeI (product 1) and XbaI (product 2) and then ligated to produce a sequence much like:
OmpABgeginning-XbaISpeIFusionSite-Randomer(15 nt)-Rest of OmpA with appropriate BB sites
- The first procwedure I utilized involved digesting and ligating the inserts together to form a completet insert -- OmpA modified to include a strep or his tag at the extracellular portion of ompA
- The second strategy depended ona 3 part ligation involving the cip treated vector(already digested with e/p, and the two parts of modified ompA -- portion 1(e/s) and 2(x/p).
- While all these attempts gave us transformant colonies, and the colony pcr results (see below) were promising, the sequencing results imply something else all together. Pls. note that we did not sequence all the colonies.
- did a cpcr targetting the part 5 IU had inserted into psb1ak3
and the lanes are:
lane 1: ladder
lane 2: 5jc8
lane 3: 5j8s/p
lane 4: 5j3
Lane 5: 5j3s/p
Lane 6: 85j
Lane 7: 65j
Lane 8: 55j
Lane 9: 75j
j refers to the plasmid that contained the j04500 and part 5
- This week (of 8/10), I decided to enhance my efforts in using the OmpA gene with an NheI site at the psn where we expect to have a extracellular addition to native OmpA. There are 2 main strategies involved:
- (i)whence we will use a double digestion N/P and then fuse in X/P-ended inserts for 2 randomers, strep2 and 6xHis
- (ii)I got good results for this -- they are indicative of a successful transformation in that the unligated, yet digested plasmids had no colonies whatsoever. see below:
Negaive control
Experiment Proper -- note the white colonies
