Lab 208

Main purpose

• PCR for SPL and Ref (reference promoter)
• PCR for AMO with USER endings
• High yield plasmids purification of cycAX, Sec2, TAT2-1, TAT3-2 and TAT3-1a.
• Gel purification of AMO, araBAD promoter and Nir2.
• USER reaction on araBAD inducible promoter and pZA21 without native promoter containing GFP Sf
• more PCR

Who was in the lab

Kristian, Gosia, Julia, Henrike

Procedure

PCR for SPL and Ref (reference promoter)

template: pZA21 with RFP;

primers for SLP: 52a, 52b1; primers for Ref: 52a, 52b2;

temperature: 53C, time: 3:00;

Samples:

1 -> SLP + DMSO

2 -> SLP + betaine

3 -> Ref + DSMO

4 -> Ref + betaine

5 -> SLP Negative control + DMSO

6 -> Ref Negative control + betaine

PCR for AMO with USER endings

Midiprep

Purifying plasmids with high yield for sequencing of the samples cycAX, Sec2, TAT2-1, TAT3-2 and TAT3-1a.

Gel purification

Extracted from gel and purified AMO extraction fragment, araBAD promoter and Nir2 extraction fragment. Nir2 was treated according to protocol for fragments bigger 4kb, the others as fragments between 100bp and 4kb.

USER reaction

Performed USER ligation on araBAD inducible promoter and pZA21 without native promoter containing GFP Sf respective RFP following the standard protocol (I assume).

more PCR

Results

Gel on yesterdays PCR

ara, SPL is from yesterdays PCR with 5% DMSO, Nir and the sample numbers is also from yesterday, here the composition can be seen.

• ara
• ara
• SPL
• SPL
• Nir 0 MgCl2
• Nir 1uL MgCl2
• Nir 5uL MgCl2
• Nir 20uL MgCl2
• 12 - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, x7 polymerase
• 13 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
• 14 - Nir1 with USER primers, primers 39a, 39b, x7, template - Nir purified from gel
• 15 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
• 16 - check Nir1 with primers for NirM+S, primers 11a, 44, template - Nir purified from gel, Phusion polymerase
• 7 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
• 8 - Nir2, extraction PCR, primers 42a, 42b, cells in water, Phusion polymerase
• 9 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase
• 10 - Nir2, extraction PCR, primers 42a, 42b, cells in water, x7 polymerase

not on the gel: 11 - Nir2, extraction PCR, primers 42a, 42b, cells in water, plus MgCl2 additive, Phusion polymerase

Decided to purify ara and Nir2 (from 12, 7, 8, 9 and 10) and analyse sample 11 (Nir2).

Purification gel

• 1 kb ladder
• ara
• ara
• 12
• 7
• 8
• 9
• 10
• 11
• Nir colony PCR X7
• 1 kb

Lab 115

Main purpose

Run Experiment 2 anaerobically in order to characterize the behavior of P. aeruginosa

Who was in the lab

Ariadni, Helen

Procedure

Adjusting the temperature at 37 degrees and calibrating the probes as described in Appendix 5.

Following the protocol Experiment 2

Changing the steps :

6. 2 ml of the overnight culture in 100 ml of medium.

7. Grow the cells for about 4 hours where the OD was measured ...... in 500 nm wavelength setup instead of 600 nm.

8. We didn't cool down the centrifuge because the experiment had to be done in 37 degrees.

11. The volume was 140 ml instead of 200 ml.

12. The OD was measured 0.1195 instead of 0.3.

19. Add 0.5 ml of nitrite solution and continue by adding 1 ml after 10 minutes then we took 1.8 ml of sample, we added then 0.5 ml and afterwards when there was any change in the curve we spiked with 0.9 ml of nitrite. We took 2.3 ml for sample in the end and another 2 ml for OD measurement where OD=0.1105.

The temperature in the end was 40.5 degrees C and not 37.

Results

Colorimetric results

Ammonium

Measuring range 2-75 mg/L NH₄-N

start point- signal <2 mg/L

middle point- signal <2 mg/L

end point- signal <2 mg/L but 1.8 mg/L

Nitrate

Measuring range 1-25 mg/L NO₃-N

start point- signal <1 mg/L

middle point- signal <1 mg/L

end point- signal 0.4 mg/L

Nitrite

Measuring range 0.02-1 mg/L NO₂-N

start point- signal 0.07 mg/L

middle point- signal 0.57 mg/L after X10 dilution

end point- signal 0.47 mg/L after X10 dilution

N₂O measurements about P. aeruginosa

Conclusion

We were not able to see a response.

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