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Main purpose

• miniprep of biobrick transformants from 10-07-2013
• PCR with USER primers to amplify cytochromes, AMO, HAO and Nir genes
• gel analysis of PCR products from Nir operon, AraBAD promoter and RFP
• USER reaction of RFP and pZA21 (with native promoter) and transformation of E. coli cells
• Gel on todays PCR-reactions.

Who was in the lab

Henrike, Julia, Kristian, Jakob, Gosia

Procedure

Plasmid isolation of biobricks from transformants made on 10-07-2013

Isolation was performed according to protocol given in the kit PowerPrep HP Plasmid Miniprep Kit

Amplification of cytochromes, AMO, HAO and Nir genes

Each PCR reaction was performed in triplicate.

Samples are named:

• 1, 2, 3 -> cytochromes
• 4, 5, 6 -> AMO
• 7, 8, 9 -> HAO
• 10, 11, 12 -> Nir

PCR reaction mix was done according to standard procedure.Primers and templates used for samples are as follows:

• 1, 2, 3 -> cytochromes, primers 16a, 16b, template CYC isolated by colony PCR from Nitrosomonas europea
• 4, 5, 6 -> AMO, primers 17a, 17b, template AMO isolated by colony PCR from N. europea
• 7, 8, 9 -> HAO, primers 18a, 18b, template HAO isolated by colony PCR from N. europea
• 10, 11, 12 -> Nir, primers 15a, 15b, template - 2uL of liquid culture of "Pseudosomonas"

PCR programs based on standard program with differences in annealing temperature and elongation time as follows:

• 1, 2, 3 -> cytochromes - 57°C, 1:30 min
• 4, 5, 6 -> AMO - 54°C, 3 min
• 7, 8, 9 -> HAO - 59°C, 9 min
• 10, 11, 12 -> Nir - 59°C, 9 min

Gel analysis of PCR products from Nir operon, AraBAD promoter and RFP

1 % Agorase Gel (Nir operon)

Wells

• 1: ladder 100bp NEB
• 2: RFP
• 3: RFP
• 4: AraBAD promoter
• 5: AraBAD promoter
• 6: AraBAD promoter
• 7: RFP
• 8: Nir
• 9: Nir
• 10: Nir
• 11:Nir
• 12: Nir
• 13: Nir
• 14: Nir
• 15: Ladder 1 kb

USER reaction of RFP and pZA21 (with native promoter) and transformation of E. coli cells

USER reaction was performed according to standard protocol.

USER mix:

• USER enzyme 1 uL
• NEB 4 buffer 0,5 uL
• 10x BSA 0,5 uL
• backbone pZA21 1uL

• Sample 1 - 3 uL of USER mix + 7 uL of RFP
• Sample 2 - Negative control, USER mix + 7 uL water

USER reaction was performed in PCR machine with programm: 37C for 40 min and then 25C for 30 min.

To 100uL of chemically competent E. coli cells 10 uL of USER mix after USER reaction was added.

Incubation of cells on ice for 30 minutes, heat shock at 42C for 90 sec. and 5 min on ice. Addition of SOC medium and 2 hours of incubation at 37C. Plating on plates with LB medium with Kanamycin, overnight incubation at 37C.

Gel 2; on todays PCR-products for USER-cloning (cycAX, AMO, HAO, Nir)

Wells

• 1: ladder 100bp NEB
• 2: cycAX, tube 1
• 3: cycAX tube 2
• 4: cycAX tube 3
• 5: AMO tube 4
• 6: AMO tube 5
• 7: AMO tube 6
• 8: HAO tube 7
• 9: HAO tube 8
• 10: HAO tube 9
• 11: Nir tube 10
• 12: Nir tube 11
• 13: Nir tube 12
• 14: Ladder 1 kb

Colony PCR to extract the Nir operon directly with USER-primers

Conclusion

We have USER fragments for RFP and AraBAD which was verified with the first gel but Nir operon is another story. The Nir primers seems to make lots of unspecific products. We tried to do the colony PCR with the USER primers directly and this gave fragments longer than expected but not with unspecific product though. We will try and transform this fragment into E.coli and see what will happen and then we will send the construct to sequencing so that we can see whats causing the elongation of the fragment.