Template:Groningen/Labjournal/PatpI-in-pSB1C3

P_AtpI in pSB1C3 (BBa_K1930005)

The promoter P_AtpI has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 min of the germination of B. subtilis (Sinai et al. 2015). atpA gene is part of an operon atpI-atpB-atpE-atpF-atpH-atpA-atpG-atpD-atpC, therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen. The region was checked for the binding of sigma factors and transcription factors with DBTBS. No binding factors were found with the highest significance level. In our project we wanted to find a constitutive promoter for our ciprofloxacin resistance casette. In the following part we put the promoter P_AtpI in the pSB1C3 backbone to make it available to other iGEM teams.

PCR

Experiment:

06/10/16: The promoter P_AtpI was amplified (see PCR protocol) from the ciprofloxacin resistance cassette BioBrick BBa_K1930004) with the primers pATPI+prefix and pATPI+suffix (primer sequences can be found here). The correct size of 372 bp for the PCR product was checked with DNA electrophoresis. The PCR product was stored at 4°C.

PCR mixture:

50 μl PCR assay was performed according to the following protocol.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Figure 1. DNA electrophoresis with amplified P_AtpI promoter sequence 372 bp in size.

Conclusion:

The P_AtpI promoter was successfully amplified with prefix and suffix from the plasmid.

Procedure after gel validation:

PCR product was subsequently cleaned with PCR Purification Kit – Jena Bioscience.

Restriction digestion

Experiment:

07/10/16: The P_AtpI was cut with EcoRI and PstI. The backbone pSB1C3 (BBa_J04450) was digested with the same enzymes. This construct is carrying RFP reporter therefore it was used for easier screening after transformation. You could see self-ligations as red colonies and the correct ones as white ones.

RD mixture:

30 μl RD assay was performed according to the following protocol.

DNA Electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Figure 2. DNA electrophoresis of EcoRI and PstI digested pSB1C3 - BBa_J04450. The RFP insert is 1069bp and the pSB1C3 backbone is 2019 bp.

Conclusion:

The digestion was successful because bands for both expected fragments could be seen on the gel, namely RFP insert is 1069bp and the pSB1C3 backbone is 2019 bp.

Procedure after gel validation:

The upper band of 2000 bp was cut out from the gel and DNA was extracted by Gel extraction kit (Nucleospin). The PCR product was not checked on the gel after the digestion but immediately cleaned up with (CR Purification Kit – Jena Bioscience.

Ligation

Experiment:

07/10/16: The EcoRI, PstI cut pSB1C3 backbone was ligated with the EcoRI, PstI cut promoter P_AtpI.

Ligation mixture:

20 μl ligation assay was performed according to the following protocol.

Transformation

Experiment:

08/10/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the protocol. Cells were plated on 50 μg/ml chloramphenicol LB agar to select the correct constructs. The next day colonies were picked to perform colony PCR to find the correct constructs with the primers pATPI+prefix and pATPI+suffix (primer sequences can be found here).

Figure 3. E. coli Top10 transformed with P_AtpI in pSB1C3.

PCR mixture:

25 μl PCR assay was performed according to the following protocol.

PCR set-up:

95ºC	2:00 min
95ºC	30s	(30X)
60ºC	30s	(30X)
72ºC	1:30 min	(30X)
72ºC	2:00 min
10ºC on hold

DNA electrophoresis:

For detailed information on how to prepare and run agarose gel see following protocol.

Figure 4. Result of the colony PCR. Samples from colony 1-5 are showing the correct size of the promoter P_AtpI of 372 bp. C - is the water control.

Conclusion:

The transformation of P_AtpI in pSB1C3 to E. coli Top10 was successful.

Validation

Experiment:

09/10/16: Grown cultures of E. coli Top10 with the construct were used to make glycerol stocks and plasmid isolation was performed (see Fast-n-Easy Plasmid Mini-prep kit). Firstly, concentration of the plasmids obtained was measured on Nanodrop. Secondly, plasmids were sent for sequencing and then stored at -20°C.

Sequencing:

The plasmid P_{AtpI in pSB1C3 from colonies 1 and 2 was sent for sequencing with the primers VF2 and VR, see primer list.}

Figure 6. Sequencing result for P_AtpI in pSB1C3 from colony 1 with VR.

Figure 5. Sequencing result for P_AtpI in pSB1C3 from colony 1 with VF2.

Conclusion:

The sequencing result proofed the successful integration of the P_AtpI promoter in the pSB1C3. Another BioBrick was obtained!

Experiments

For further experiments with this BioBrick see: MIC value experiment.