Difference between revisions of "Part:BBa K3034006"
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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. | 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 Abs600==== |
− | Moreover, we defined the | + | Moreover, we defined the Relative Abs600 to represent the resistance ability of our part. The higher the Relative Abs600, the stronger the resistance. When the value was less than 1, it meant that the growth was suppressed. |
− | [[File:T-UESTC-China- | + | [[File:T-UESTC-China-relative_abs600.png|500px|center]] |
− | It could be seen intuitively from fig.4 that our ''E.coli'' DH5α carrying ' | + | We chose a significant concentration 1mg/L to represent the growth situation(fig.4). It could be seen intuitively from fig.4 that our ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034006'>BBa_K3034006</a></html> 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. |
− | + | ||
− | [[File: | + | [[File:T-UESTC-China-new_relative.png|700px|thumb|center|'''fig.4''' The Relative Abs600 of Wild-Type ''E.coli'' DH5α and ''E.coli'' DH5α(with <html><a href='https://parts.igem.org/Part:BBa_K3034006'>BBa_K3034006</a></html>).]] |
====Conclusions==== | ====Conclusions==== |
Revision as of 06:59, 11 October 2019
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 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.
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 Abs600. Then we added gradient concentrations of CIP and measured Abs600 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).
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).
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 Abs600
Moreover, we defined the Relative Abs600 to represent the resistance ability of our part. The higher the Relative Abs600, the stronger the resistance. When the value was less than 1, it meant that the growth was suppressed.
We chose a significant concentration 1mg/L to represent the growth situation(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.
![](/wiki/images/a/a8/T-UESTC-China-new_relative.png)
Conclusions
- 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.
- The resistance of wild-type E.coli DH5α is stronger than wild-type E.coli Top10.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 36
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]