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.

Usage and Biology

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]

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]