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

Minimal Inhibitory Concentration (MIC) is the lowest concentration of antibiotic which prevents growth of bacteria. 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 (see Figure 1 and 2). 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. This is a significant improvement antibiotic tolerance (approximately 10x more resistance for E. coli and 2.3x more resistance for B. subtilis).

Results:

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

Figure 2 – Growth of E. coli Top10 carrying qnrS1 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. 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. 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.

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 3 – B. subtilis 168 growth curve at 37°C.

Figure 4 – Growth of B. subtilis 168 in the presence of ciprofloxacin (25 nM- 2,500 nM).

Figure 5 – Growth of B. subtilis 168 in the presence of ciprofloxacin (100 nM – 200 nM).

Figure 6 – B. subtilis - 130, 160, 170 nM 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) - Colony 1 With Ciprofloxacin</figcaption> </figure>

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<figcaption>Figure 15 – Growth of B. subtilis 168 (with qnrS1) - Colony 2 With Ciprofloxacin</figcaption> </figure>

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<figcaption>Figure 16 – Growth of B. subtilis 168 (with qnrS1) - Colony 3 With Ciprofloxacin</figcaption> </figure>

The transformation to B. subtilis was performed according to the following protocol: [http://2016.igem.org/Team:Groningen/Labjournal#transform-b-subtilis Transformation into the B. subtilis]. Colonies were selected on LB agar plates with 5 μg/ml chloramphenicol.

Figure 1. 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 [http://2016.igem.org/Team:Groningen/Labjournal#Starch-test Starch test].

Figure 2. 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 3 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. [http://2016.igem.org/Team:Groningen/Experiments" See here]

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<p>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 14).

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<figcaption>Figure 14 – Growth of B. subtilis 168 (with qnrS1) - Colony 1 With Ciprofloxacin</figcaption> </figure>

<figure class="left flone"> <img src="" />

<figcaption>Figure 15 – Growth of B. subtilis 168 (with qnrS1) - Colony 2 With Ciprofloxacin</figcaption> </figure>

<figure class="right flone"> <img src="" />

<figcaption>Figure 16 – Growth of B. subtilis 168 (with qnrS1) - Colony 3 With Ciprofloxacin</figcaption> </figure>

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]