Template:Groningen/Labjournal/Cipro-in-pSB1C3

Ciprofloxcacin cassette in pSB1C3

To design a qnrS1 resistance cassette biobrick we designed a gBlock that contains the Bacillus subtilis promoter PatpI, 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 PatpI+RBS+QnrS1+Ter2, see plasmid map.

Amplification of the qnrS1 sequence

Experiment:

The sequence of the qnrS1 gene was amplified from the gBlock qnrS1 E. coli ordered from IDT (link to sponsors). Primers used for the amplification were F-qnrs1 e.coli and R-qnrs1 e.coli. See the primer listhere.

PCR mixture:

50 μl PCR assay was performed according to the following protocol: PCR (Q5® High-Fidelity)

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:

The PCR samples were loaded on a 1 % agarose gel. For detailed information on how to prepare and run agarose gels see following protocol DNA electrophoresis.The amplified qnrS1 sequence has a size of 1194 bps.

Conclusion:

Figure 1. Gel electrophoresis of the PCR product.

The PCR of the qnrS1 sequence from the IDT gBlock was successful. It was verified by DNA electrophoresis (Figure 1). A band with the correct size of 1194 bps could be seen.

Procedure after gel validation:

The PCR product was subsequently cleaned with the Gel extraction kit Gel extraction (Agarose Gel Extraction Kit – Jena Bioscience).

Restriction Digestion:

Experiment:

28/09 The qnrS1 gene should be cloned into the biobrick shipping vector BBa_pSB1C3. For this case the vector with BBa_J04450 was used. It consists of a red fluorescent protein in the shipping vector BBa_pSB1C3.

The qnrS1 gene and BBa_J04450 were cut with the restriction enzymes EcoRI and PstI.

RD mixture:

The restriction was performed in 20 μl RD assay according to the restriction digestion assay Restriction digestion (FastDigest enzymes).

DNA Electrophoresis:

The restricted samples were analyzed on a 1 % agarose gel. For detailed information on how to prepare and run agarose gels see following protocol DNA electrophoresis.

Bands of the sizes 2029 bps for the vector and 1176 for the gene were expected.

Figure 2 Gelelectrophoresis of EcorI and PstI digested pSB1C3 + 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 digested samples were cut out from the gel and the DNA was extracted using the Nucleospin Gel Extraction Kit Gel extraction (Agarose Gel Extraction Kit – Jena Bioscience)

Ligation:

Experiment:

28.9.2016: After restriction digestion ligation was performed. 6 µl qnrS1 insert DNA were ligated to 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 ligation protocol .Ligation

Transformation:

Experiment:

28/9 After ligation 2 µl of ligation were transformed into E. coli Top10. The transformation was plated on plates containing 50 µg/ml chloramphenicol. The transformation was performed according to the transformation protocol

Colony PCR:

Experiment:

29/09 To analyze the success of the transformation 12 samples were picked and and a colony PCR was performed according to the colony PCR protocol Colony PCR. The primers F-qnrs1 e.coli and R-qnrs1 e.coli were used. here

DNA electrophoresis:

The colony PCR was loaded on a 1 % agarose gel. For detailed information on how to prepare and run agarose gels see following protocol. DNA electrophoresis

Conclusion:

The colony PCR did not bring clear results about the efficiency of the transformation (Figure 3).

Control restriction digestion:

Experiment:

30/9 To further analyze the success of the transformation four colonies were grown as overnight cultures (started on 29.09.2016) in LB and 50 µg/ml chloramphenicol and were digested using the enzymes EcoRI and PstI according to the restriction digestion protocol Restriction digestion (FastDigest enzymes)

DNA electrophoresis:

The digested samples were loaded on a 1 % agarose gel. For detailed information on how to prepare and run agarose gels see following protocol DNA electrophoresis.

Conclusion:

Two out of the four digested samples showed bands of the expected sizes 2029 bps for the vector and 1184 for the gene could be seen (Figure 4).

Validation:

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 {{primerlist}} and {{sequencing}}. 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 ).Thus the qnrS1 resistance cassette could successfully be integrated into the iGEM shipping backbone pSB1C3.