Part:BBa_K1930004

Ciprofloxacin resistance cassette

This part is a ciprofloxacin resistance cassette. It is containing a P_AtpI constitutive promoter, ciprofloxacin resistance gene qnrS1 and double terminator.

The promoter P_AtpI (BBa_K1930005) is a constitutive promoter which has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 minutes of the germination of B. subtilis. atpA gene is part of an operon (atpI-atpB-atpE-atpF-atpH-atpA-atpG-atpD-atpC), therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen.

Ciprofloxacin resistance gene qnrS1 is found naturally in E. coli, and other Gram-negative strains. 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.

Double terminator (BBa_B0015) is the most commonly used terminator. For more information see here: https://parts.igem.org/Part:BBa_B0015.

The MIC value for the wild-type strains was found to be between 100-130 nM for Escherichia coli and 160 - 170 nM for B. subtilis. MIC value for E. coli Top10 and B. subtilis 168 carrying the qnrS1 ciprofloxacin resistance gene was determined to be between 1000 - 2000 nM, and 400 - 500 nM, respectively.

Usage and Biology

MIC test – Ciprofloxacin, wild-type E. coli Top10

Minimal Inhibitory Concentration (MIC) is the lowest concentration of antibiotic which prevents growth of bacteria. The MIC value for the wild-type strain was found to be between 100-130 nM for Escherichia coli (see Figure 1). Ciprofloxacin MIC test was carried out on wild-type E. coli Top10 to determine the concentration of the antibiotic that this strain could tolerate.

Results:

Figure 1. Growth of wild-type *E. coli* Top10 in the presence of ciprofloxacin

MIC and MBC tests – Ciprofloxacin, Wild-Type B. subtilis 168

Minimal Inhibitory Concentration (MIC) is the lowest concentration of antibiotic which prevents growth of bacteria. Minimal Bactericidal Concentration (MBC) is the lowest concentration that kills bacteria. Ciprofloxacin MIC and MBC tests were carried out on wild-type Bacillus subtilis 168 to determine the concentration of the antibiotic that this strain could tolerate.

Two tests were carried out with ciprofloxacin. The growth of B. subtilis 168 in a 96-well plate with a range of ciprofloxacin concentrations were monitored for 18 and 24 hours. The positive control was used to construct the growth curve (see Figure 2). For the first test a range of 25 nM to 2,500 nM was used. Results showed that the MIC was somewhere between 100 nM and 200 nM (see Figure 3). The second time a range of 100 nM to 200 nM was tested to determine the MIC more precisely. Results showed that the MIC of ciprofloxacin on B. subtilis 168 was 160 - 170 nM (see Figures 4 and 5).

Now that we obtained the MIC value, we determined the MBC. 10 µL of 160 nM, 170 nM and 180 nM cultures from the 96-well plate were plated on LB agar in duplo. These were incubated overnight at 37°C. Only the 180 nM plate had no growth, so we take that to be the MBC.

Results:

Figure 2. *B. subtilis* 168 growth curve at 37°C

Figure 3. Growth of *B. subtilis* 168 in the presence of ciprofloxacin (25 nM- 2,500 nM)

Figure 4. Growth of *B. subtilis* 168 in the presence of ciprofloxacin (100 nM – 200 nM)

Figure 5. *B. subtilis* - 130, 160, 170 nM ciprofloxacin

MIC tests – Ciprofloxacin, E. coli Top10 (carrying qnrS1) and B. subtilis 168 (carrying qnrS1)

MIC tests were carried out as done previously. MIC value for E. coli Top10 and B. subtilis 168 carrying the qnrS1 ciprofloxacin resistance gene was determined to be between 1000 - 2000 nM, and 400 - 500 nM (see Figure 6 and 7). This is a significant improvement in antibiotic tolerance (approximately 10x more resistance for E. coli and 2.3x more resistance for B. subtilis).

Results:

Figure 6. Growth of *E. coli* Top10 carrying *qnrS1* in the presence of ciprofloxacin

A MIC test was carried out on three colonies of B. subtilis 168 transformed with the qnrS1 resistance gene. Colony 1 was the most resistant, with a MIC between 400 and 500 nM (see Figure 7).

Figure 7. Growth of *B. subtilis* 168 (with *qnrS1*1) - Colony 1 With Ciprofloxacin

Transformation of ciprofloxacin resistance cassette into the B. subtilis 168

Experiment:

The transformation into the B. subtilis 168 was performed according to the following protocol. Colonies were selected on LB agar plates with 5 μg/ml chloramphenicol.

Figure 8. *B. subtilis* after transformation with ciprofloxacin resistance cassette.

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. See Starch test.

Figure 9. Starch test. Colonies without a clear halo are positive for integration.

Conclusion:

The integration of the ciprofloxacin resistance cassette was successful.

Validation:

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 10 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 here.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 536
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Contribution

Group: iGEM Team UESTC-China 2019
Author: Qiuyun Huang, Zixin Wang

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

While testing this part, we found that the resistance effect couldn’t meet our requirements. The growth situation of E.coli DH5α was not ideal in the presence of 1mg/L CIP.

To ensure the growth and the function of our E.coli DH5α in a high concentration of CIP, we added a strong promoter BBa_J23119 and RBS BBa_K1725309 to the upstream of qnrS1, and we changed the terminator to BBa_B1006, to construct our part BBa_K3034006(fig.1).

fig.1 The Framework of BBa_K3034006.

MIC tests-Ciprofloxacin, E.coli DH5α carrying BBa_K3034006 and wild-type E.coli DH5α

Experiment

In addition, 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). And the MIC value for E.coli Top10 carrying qnrS1 was determined to be between 1uM-2uM (0.33mg/L-0.66mg/L). Regarding the resistance ability, our engineered E.coli DH5α was 30 times stronger, so it met our requirements well.

fig.2 Growth of E.coli DH5α(with BBa_K3034006) 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 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 BBa_K3034006. Thereout it verified that our part was workable.

Inhibition ratio

Moreover, we defined the inhibition ratio to represent the resistance ability of our part(fig.4). The higher the inhibition ratio, the weaker the resistance.

It could be seen intuitively from fig.4 that our E.coli DH5α carrying BBa_K3034006 showed significant resistance under 0.15mg/L-1mg/L CIP in the first three hours. It even growth better than the negative group under 0.3mg/L CIP. As time went on, resistance effect of qnrS1 was gradually weakening.

fig.4 The Inhibition Rate of Ciprofloxacin on Wild-Type E.coli DH5αand E.coli DH5α(with BBa_K3034006).

Conclusions

Our improvement of this ciprofloxacin resistance cassette (BBa_K1930004) is successful. Furthermore, our new part has a more ideal result and it meets our needs.
The resistance of wild-type E.coli DH5α is stronger than wild-type E.coli Top10.
Resistance of ciprofloxacin: E.coli DH5α carrying BBa_K3034006 > wild-type E.coli DH5α > E.coli Top10 carrying BBa_K1930004 > wild-type E.coli Top10.