Difference between revisions of "Template:Groningen/Labjournal/Cipro-in-K823023"

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:

DNA Electrophoresis:

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

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 PCR product and BBa_K823023 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.

Conclusion:

The digestion of BBa_K823023 showed the correct bands on the gel. 6,000 bp backbone for the backbone and the 1000 bp for the RFP insert. Therefore the cloning procedure could proceed.

Procedure after gel validation:

Digested sample of the backbone ~6,000 bp were cut out from the gel and DNA was extracted by Agarose gel extraction kit (Jena Bioscience) (see protocol).

The digestion of the qnrS1 PCR product was immediately cleaned with the kit (PCR Purification Kit – Jena Bioscience).

Ligation

19/10 In this ligation the EcoRI, PstI cut gene qnrS1 and the vector BBa_K823023 were combined.

Ligation mixture:

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

Transformation

Experiment:

29/09/16: The ligation mix was heat shock transformed to competent Top10 E. coli cells following the protocol. Cells were plated on 100 μg/ml ampicillin LB agar to select for the correct constructs. On the next day colonies were selected to perform colony PCR in order to find the correct constructs using the primers F-qnrs1 E.coli and R-qnrs1 E.coli. Primer sequences can be found here.

PCR mixture:

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

PCR set-up:

DNA electrophoresis:

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

Conclusion:

The transformation of qrnS1 (ciprofloxacin resistance cassette) in BBa_K823023 to E. coli Top10 was successful.

Validation

Experiment:

To test if the construct would make B. subtilis resistant to ciprofloxacin, the construct qrnS1 in BBa_K823023 was transformed into the B. subtilis 168 tpr+.

Experiments

Experiment:

13/10/16: The transformation to B. subtilis was performed according to the following protocol. Colonies were selected on LB agar with 5 μg/ml chloramphenicol.

14/10/16: Colonies were streaked out on agar with starch to perform the starch test, which verifies the integration in the amyE locus in the genome of B. subtilis. Integration check: Starch test

Conclusion:

The integration of the ciprofloxacin resistance cassette was successful.

As a first check on the functionality of the ciprofloxacin resistance cassette, we grew B. subtilis colonies from the starch test with ciprofloxacin. As a control they were also grown with chloramphenicol, the resistance on the backbone of the integration vector. Figure 7 shows the result for 3 different colonies (tubes indicated with 1 - 3). The tubes marked with Cm is the control with chloramphenicol, which shows growth for all three colonies. The tubes marked with Cipro were grown with ciprofloxacin. Colonies 2 and 3 showed growth, whereas colony 1 did not grow. Seems like the resistance cassette is working. To further explore if the ciprofloxacin cassette is functional in B. subtilis, a MIC value test was performed. See link.