Sambu/notebook

Stuff done thus far:

• Did this experiment to verify that OD and cell dilution factors were linearly related>

.

• 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:

1. Denatured for 2 mins as opposed to initial 10 mins
2. Used fresh dilutions of primers (final conc. was 10 uM)
3. 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