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

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 also our primer list

PCR mixture:

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

PCR set-up:

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:

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 PCR product and BBa_K823023 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 sample of BBa_K823023 was analyzed on a 1 % agarose gel. For detailed information on how to prepare and run agarose gels see following protocol DNA electrophoresis.

Conclusion:

The digestion of K823023 showed the correct bands on the gel for the 6000 bp backbone and the 1000 bp RFP insert. Therefore the cloning procedure could proceed.

Procedure after gel validation:

Digested sample of the backbone 6000 bp were cut out from the gel and DNA was extracted by Gel extraction kit Gel extraction (Agarose Gel Extraction Kit – Jena Bioscience)

The digestion of the qnrS1 PCR product was immediately cleaned with the kit DNA Clean-up (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:

The ligation took place in a 20 µl assay according to the ligation protocol Ligation

Transformation:

Experiment:

19/10. The ligation mix was heat shock transformed to competent Top 10 E.coli following the protocol Transformation of E. coli (Standard protocol) Cells were plated on 100 μg/ml ampicillin to select for the correct construct. The next day colonies were selected to perform colony PCR in order to find the correct construct Using the primers F-qnrs1 e.coli and R-qnrs1 e.coli. Primers can be found here primer list.

PCR mixture:

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

PCR set-up:

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:

The transformation of qrnS1 in BBa_K823023 to E.coli Top 10 was successful.

Validation:

Sample was not sent for sequencing.

Experiment:

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

13/10 Grown cultures of E.coli Top10 with the construct were used to obtain Glycerol stocks Glycerol stock and plasmid isolation was performed Plasmid mini-prep (Fast-n-Easy Plasmid Mini-Prep Kit - Jena Bioscience). The concentration of the plasmids obtained was measured on Nanodrop. Plasmids and then stored at -20°C.

Transformation

Experiment:

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

Validation

Experiment:

14/10 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 ciprofloxcancin resistance cassette was successful.

Experiments

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. Colonie 2 and 3 show growth, whereas colony 1 does 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.