Template:Groningen/Labjournal/Cipro-in-pSB1C3

Ciprofloxacin resistance cassette in pSB1C3 (BBa_K1930004)

To design a qnrS1 resistance cassette BioBrick we designed a gBlock that contains the Bacillus subtilis constitutive promoter P_AtpI, which is active from a very early stage of germination and includes a ribosome binding site. The gBlock also contains the original qnrS1 gene sequence from E. coli, the double terminator (BBa_B0015) from iGEM as well as the prefix and suffix for BioBricks. In summary the ciprofloxacin cassette consists of the following parts P_AtpI+RBS+qnrS1+2TER, see plasmid map below.

PCR

Experiment:

The sequence of the qnrS1 gene was amplified from the gBlock qnrS1 E. coli ordered from IDT. Primers used for the amplification were F-qnrs1 E.coli and R-qnrs1 E.coli (primer sequences can be found here).

PCR mixture:

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

PCR set-up:

95ºC	2:00 min
95ºC	30s	(12X)
60ºC	30s	(12X)
72ºC	1:30 min	(12X)
72ºC	2:00 min
10ºC on hold

DNA Electrophoresis:

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

Figure 1. Gel electrophoresis of the PCR product.

Conclusion:

The PCR of the qnrS1 sequence from the IDT gBlock was successful. It was verified by DNA electrophoresis. A band with the correct size of 1,194 bp could be seen.

Procedure after gel validation:

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

Restriction digestion

Experiment:

28/09/16 The qnrS1 gene should have been cloned into the pSB1C3. For this case the vector BBa_J04450 was used. The qnrS1 gene and BBa_J04450 were cut with the restriction enzymes EcoRI and PstI.

RD mixture:

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

DNA Electrophoresis:

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

Bands of the sizes 2,029 bp for the vector and 1,176 bp for the gene were expected.

Figure 2. DNA electrophoresis of EcoRI and PstI digested pSB1C3 - BBa_J04450. The RFP insert is 1,069 bp 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 1,069 bp and the pSB1C3 backbone is 2,019 bp.

Procedure after gel validation:

The digested samples were cut out from the gel and the DNA was extracted using the Agarose gel Extraction Kit (Jena Bioscience).

Ligation

Experiment:

28/09/16: After restriction digestion ligation was performed. 6 µl qnrS1 insert DNA were ligated to 4 µl pBS1C3 vector DNA. The ligation took place for 2 h at room temperature.

Ligation mixture:

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

Transformation

Experiment:

28/09/16 After ligation the ciprofloxacin resistance cassette in pSB1C3 was transformed into E. coli Top10 cells. The transformation was plated on plates containing 50 µg/ml chloramphenicol. The transformation was performed according to the transformation protocol.

29/09/16: To analyze the success of the transformation 12 samples were picked and a colony PCR was performed according to the following protocol. The primers F-qnrs1 E.coli and R-qnrs1 E.coli were used. Primer sequences can be found here.

DNA electrophoresis:

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

Conclusion:

The colony PCR did not bring clear results.

Experiment:

30/09/16: To further analyze the success of the transformation four colonies were grown as overnight cultures (started on 29/09/16) in LB and 50 µg/ml chloramphenicol. On the next day plasmid isolation was performed and plasmids were digested using the enzymes EcoRI and PstI according to the following restriction digestionprotocol.

DNA electrophoresis:

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

Conclusion:

Two out of the four digested samples showed bands of the expected sizes 2,029 bps for the vector and 1184 for the gene could be seen.

Validation

Experiment:

The two samples that showed the correct digestion pattern in the restriction digestion control were sent for sequencing with the primers VF2 and VR, see primer list. These two samples showed the correct sequence (Figure 5.1-5.4, 5.1 mutant 1 forward, 5.2: mutant 2 forward, 5.3: mutant 1 reverse, 5.4: mutant 2 reverse ).

Experiments

For further experiment see the construction of the plasmid ciprofloxacin resistance cassette in BBa_K823023.