To design a ciprofloxacin resistance cassette 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. qnr genes code for pentapeptide repeat proteins. These proteins reduce susceptibility to quinolones by protecting the complex of DNA and DNA gyrase enzyme from the inhibitory effect of quinolones. Finally, this gBlock contains a 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+2TER.BBa_K823023 is an available BioBrick from igem Munich 2012. It is an
integration plasmid for Bacillus subtilis, which can be used for
cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more
efficient screening after transformation. Construction of ciprofloxacin resistance cassette in BBa_K823023 integration plasmid was performed as described in the following.
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. DNA 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.
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.
Figure 2. BBa_K823023 cut with EcoRI and PstI. Backbone ~6,000
bp, RFP insert 1000 bp.
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).
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.
Figure 3. Transformation to E. coli Top10.
PCR mixture:
25 µl PCR assay was performed according to the following
protocol.
Figure 4. Result of the colony PCR. Sample indicated
with the star shows the correct sie of 1194 bp. C - is the water
control.
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.
Figure 5. B. subtilis after transformation with
ciprofloxacin resistance cassette.
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
Figure 6. Starch test. Colonies without a clear halo
are positive for integration.
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.
