Team:University College London/LabBook/Week11

Contents 1 Monday 20.08 2 Tuesday 21.08 3 Wednesday 22.08 4 Thursday 23.08 5 Tuesday 21.08 6 Wednesday 22.08 7 Thursday 23.08 8 Friday 24.08

Monday 20.08

Aim: To test how the amount of nuclease produced by W3110 E. coli cells changes over time

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). IPTG induces the lac promoter which in turn activates the transcription of nuclease.

2. Streak cells onto all plates at the same time

3. Incubate at 37°C

4. Apply hydrochloric 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 readings every 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.

The first digest was carried out in order to prepare the Upstream Part for ligation through an EcoRI-HF™ and SpeI preparative digest:

Ingredient	Amount
Upstream Part Plasmid	500 ng
EcoRI-HF	1 µl
Spel	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

Another digest was carried out in order to prepare the downstream DNA for ligation through an XbaI/PstI preparative digest:

Ingredient	Amount
Downstream Part Plasmid	500 ng
Xbal	1 µl
Pstl	1 µl
10X NEBuffer 2	5 µl
100X BSA	0.5 µl
H2O	to 50 µl

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. Readings indicated that both are proportionally related to time. The second run of the experiment will be done in the coming weeks so as to obtain data replicates in order to validate data (See Week 13).

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.

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. The PCR was carried out in Week 10 (See Experiment 9.2, Wednesday 15 August 2012)

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 with samples from an analytical digest in order 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)

Wednesday 22.08

Step 1 - Aim: To carry out a preparative digest on the tetracycline plasmid backbone, cobalt curli, terminator and nuclease in preparation of ligation.

Methods:

Step 2 - Aim: To carry out a ligation of cobalt curli and the terminator sequence and another ligation of the nuclease and terminator sequence, in preparation of further ligations. This was done by ligating the terminator sequence to the curli cluster of genes through an EcoRI-PstI ligation, and another ligation of the terminator to nuclease through an EcoRI-PstI ligation. Once success of ligation is confirmed, two subsequent ligations will be carried out to attach the ligated sequences to the promoter and RBS, and eventually the backbone. Methods:

Destination Plasmid DNA	500 ng
EcoRI-HF	1 µl
EcoRI-HF	1 µl
Pstl	1 µl
10X NEBuffer	2 5 µl
100X BSA	0.5 µl
H2O	to 50 µl

COMPONENT	20 μl REACTION)
10X T4 DNA Ligase Buffer	2 μl
Vector DNA (3 kb)	50 ng (0.025 pmol)
Insert DNA (1 kb	50 ng (0.076 pmol)
Nuclease-free water	to 20 μl
T4 DNA Ligase	1 μl

2. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

3. Gently mix the reaction by pipetting up and down and microfuge briefly.

4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

5. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).

6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.