Team:University College London/LabBook/Week11

(11.1) Monday 20.08

Aim: To test how the amount of nuclease produced by W3110 E. coli cells changes over time (and varies with the size of the colony?).

Methods: (to make in a protocol format please For Wnu cell line which has native secreted nuclease activity 1. Prepare 11-16 plates (10ml LBAgar+10ul AMP +10 ul 1M IPTG). IPTGinduces the lac promoter which in turnactivates the transcription of nuclease. 2. Streak cells onto all plates at the same time 3. Incubate at 37°C 4. Applyhydrchloric acid (HCL) to the first plate before putting in the incubator (set time as zero) 5. Take a second reading after four hours, followed by six readingsevery 3 hours, and a final three readings every two hours. 6. When the reading is taken, observe the following: a) Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours) b) Diameter of the halo that is achieved once HCL is applied c) OD from a) d) Estimate of the depth of the colony on the agar plate For BL21 cell line that has been modified to contain nuclease 7. Prepare 11-16 plates (LB Agar + CMP) 8. Streak cells onto all plates at the same time 9. Incubate at 37°C 10. Apply HCL to the first plate before putting in the incubator (set time as zero) 11. Take a second reading after four hours, followed by six readings every 3 hours, and a final three readings every two hours. 12. When the reading is taken, observe the following: a. Diameter of the colony (once the diameter of the colony is measured, pick the colony and put it to grow in LB for nine hours) b. Diameter of the halo that is achieved once HCL is applied c. OD from a) d. Estimate of the depth of the colony on the agar plate

Results: The following table shows readings of the colony and halo diameters, plus the OD, taken at different times over 28 hours.

Date Time Colony diameter Halo diameter Absorbance at 600 OD 20.08.2012 12.30pm 0 0 0 20.08.2012 16.30pm 0 0 0 20.08.2012 19.30 pm 0 0 0 20.08.2012 22.30 pm 0.5mm 1mm 0.001 21.08.2012 01.30 am 1mm 3mm 0.142 21.08.2012 04.30 am 1.5mm 7mm 0.228 21.08.2012 07.30 am 1.5mm 7.5mm 0.252 21.08.2012 10.30am 2.5mm 11mm 0.522 21.08.2012) 12.30pm 3mm 13mm 0.749 21.08.2012 14.30pm 3.5mm 14.5mm 0.844 21.08.2012 16.30pm 4mm 16mm 0.937

Conclusion: From the data collected, it is clear that colony size correlates with the nuclease produced, which is represented by the diameter of the halo diameter. Both are proportionally related to time. The second run of the experiment will be done in a period of several weeks so that we would have data replicates in order to validate data (See Week 13).

(11.2) Tuesday 21.08

Aim: To make chemically competent W3110 E. coli Cells – Day 1

Methods: Day 1 protocol. See previous experiments.

Wednesday 22.08

Aim: To make chemically competent W3110 E. coli Cells – Day 2

Methods: Day 2 protocol. See previous experiments.

Thursday 23.08

Aim: To make chemically competent W3110 E. coli Cells – Day 3

Methods: Day 2 protocol. See previous experiments.

(11.3) Tuesday 21.08

Aim: To carry out PCR clean-up to purify the Laccase gene and Curli cluster of genes achieved from PCR carried out

Method: Please refer to previous protocols The following table shows the gene which was extracted from PCR and the primers which were used for the PCR. Gene Primers used Curli CF1 - C2R Curli CF3 – C4R Curli CF1 – C4R Curli CF3 – C2R Laccase LFONE - LR1 Laccase LFTWO - REVLF2 Laccase LR1 – LFTWO IrrE STF1 – ST2R IrrE STF3 – ST4R

Results: The following diagram shows the results of a gel run to confirm plasmid identity of Curli and Laccase after PCR clean-up, with each well having 3ul of PCR purified product. In addition the table on the bottom right shows the concentration of the purified products. The samples in bold below represent the PCR reactions which where considered successful, and hence taken forward for ligation.

The following table shows a gel run with sampled of the purified IrrE gene. As can be seen from the gel, the purified material remained in the well, indicating that PCR clean-up was not successful.

Conclusion: Since PCR was unsuccessful, this was repetead (See Week 12)

11.4