Part:BBa_K3034006

Ciprofloxacin resistance cassette

This part is a ciprofloxacin resistance cassette. It contains a strong promoter J23119(BBa_J23119), RBS(BBa_K1725309), ciprofloxacin resistance gene qnrS1 (NCBI Reference Sequence: NG_050543.1, 101...757, with condon modification) and a terminator(BBa_B1006)(Fig.1).

Ciprofloxacin resistance gene qnrS1 is first found in Gram-negative strains. It's one of the plasmid-mediated quinolone resistance genes and it encodes pentapeptide duplicates of about 200 amino acids. These proteins can produce quinolone resistance through two different mechanisms of action. One is decreasing the number of quinolone target sites by reducing DNA binding to helicase or topoisomerase IV. The other one is to inhibit the binding of quinolone to enzymes by binding these proteins to helicase or topoisomerase.

Fig.1 The Framework of BBa_K3034006.

Usage and Biology

In order to let the engineered E.coli DH5α grow and play a role normally under the exist of ciprofloxacin(CIP), we constructed this ciprofloxacin resistance cassette to enhance the resistance ability of our engineered E.coli DH5α.

MIC tests-Ciprofloxacin, E.coli DH5α carrying qnrS1 and wild-type E.coli DH5α

Experiment

We would like to increase the resistance effect, so we selected DH5α chemically competent cell to do transform. We shook the bacteria fluid overnight and adjusted them to the same OD600. Then we added gradient concentrations of CIP and measured OD600 per hour using 96 wells plate.

Results

Minimal Inhibitory Concentration (MIC) is the lowest concentration of antibiotic which prevents growth of bacteria. As for our E.coli DH5α carrying qnrS1, the MIC value was found to be between 10mg/L-50mg/L (30uM-150uM) (Fig.2).

Fig.2 Growth of E.coli DH5α(with qnrS1) in the Presence of Ciprofloxacin.

In the presence of different concentrations, we selected another wild-type E.coli DH5α which didn’t carry qnrS1 as the negative control group. And we tested its growth curve over time (Fig.3).

Fig.3 Growth of Wild-Type E.coli DH5α in the Presence of Ciprofloxacin.

We discovered that MIC value for negative group was between 0.3mg/L-1mg/L. iGEM16_Groningen(BBa_K1930004) tested the MIC value for wild-type E.coli Top10 and it was between 100nM-130nM (0.033mg/L-0.043mg/L). Comparing these two results, we found that wild-type DH5α has the higher resistance than Top10.

Besides, the MIC value for wild-type DH5α was much lower than our E.coli DH5α carrying qnrS1. Thereout it verified that our part was workable.

Relative Bacterial Density

Moreover, we defined the Relative Bacterial Density to represent the resistance ability of our part. The higher the Relative Bacterial Density, the stronger the resistance. When the value was less than 1, it meant that the growth was suppressed.

And we chose the experimental data of 1 mg/L CIP to analyze the Relative Bacterial Density defined above (Fig.4). It could be seen intuitively from Fig.4 that our E.coli DH5α carrying BBa_K3034006 grew normally in the presence of 1mg/L CIP. Meanwhile, the values of Wild-Type E.coli DH5α were all less than 1. So, we could conclude that qnrS1 conferred resistance to CIP in E.coli DH5α indeed.

Fig.4 The Relative Bacterial Density of Wild-Type E.coli DH5α and E.coli DH5α(with BBa_K3034006).

We filmed the bacterial liquid after overnight culture with 1mg/L CIP(Fig.5), and there were a significant difference between them.

Fig.5 The growth situation of Wild-Type E.coli DH5α(left) and E.coli DH5α(with BBa_K3034006)(right) .

Conclusions

1. The MIC value of E.coli DH5α carrying qnrS1 is between 10mg/L-50mg/L (30uM-150uM). And the MIC value of E.coli DH5α without qnrS1 is between 0.3mg/L-1mg/L (1uM-3uM). So this part can significantly improve the ciprofloxacin resistance of E.coli DH5α.
2. The resistance of wild-type E.coli DH5α is stronger than wild-type E.coli Top10.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 36
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]