Team:TU-Delft/Notebook/2013/09/17/

1. Restriction digestion was carried out and on gel :



2. Colont PCR was done on pT7 Lysis colonies which were transformed into BL21 yesterday:



3. Restriction digestion was carried out on:
          pBAD Receiver with S+P
          PCR Purified SUMO Peptide Tet R with X+P
          Lysis device with E+X
          pcI Ulp with E+S



4. The lysis experiment was carried out again on the pT7 Lysis transformed into BL21 plysS to check whether on inducing the pT7 promoter with IPTG, the cells lyse. The over night grown culture was diluted 1/50 around 1:50 PM. The OD was monitored till it reached 0.5 which was around 3.00 PM.
Then 30 mL of culture was taken in three different tubes and induced with 10mM, 1mM and 0.1mM concentrations.
The OD was monitored to check for death of cells :
          Time           --> 10mM           1mM           0.1mM           Control(No IPTG)
          At 3.00 PM           --> 0.54           0.54           0.54           0.54
          At 4.00 PM           --> 0.252           0.242           0.275           0.592
          At 4.20 PM           --> 0.252           0.31           0.24           0.791

The experiment proved that the lysis of cells was successful due to the fact that the cells were being lysed upon induction by IPTG.
For a better time line of the lysis experiment, we used the plate reader every ten minutes to monitor the lysis of cells.
5. Liagtions were done for :
          pBAD Receiver + SUMO peptide Tet R
          Lysis device + pSB1C3
          pBAD Ulp TT + pT7 SUMO Signiferin
6. Reinoculated pT7 Lysis in BL21 plysS for lysis experiment on plate reader.