Sambu/notebook

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

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