Test System

Introduction

In order to characterize our new antibiotic we wanted to answer three questions: 1) Is our substance active against bacteria and ideally against MRSA? 2) What is the mode of action behind the antibiotic effect? 3) Will it be active against Gyrase inhibitor resistant S.aureus? The first question can be easily examined by performing agar-diffusion assays with different bacteria. To avoid using MRSA we decided to clone the resistance providing genes into E.coli. This was accomplished by cloning SHV-1 into a vector with a L-rhamnose inducible promoter. The second question is harder to answer. Clorobiocin acts by inhibiting the ATP binding to Gyrase B. As this will most probably be also the mode of action of our new antibiotic, we cloned an aminocoumarin resistant GyrB variant from E.coli into vectors for inducible expression to use in agar-diffusion assays. The most straightforward way to test the mode of action is to purify the Gyrase and add it together with ATP to supercoiled plasmid DNA. Normally the Gyrase would relax the supercoiled DNA which could be detected on an agarose gel in a band shift. If the antibiotic inhibited the Gyrase the band shift wouldn’t be visible even after ATP addition. To answer question three we wanted to test the aminocoumarin resistant GyrB variant from S.aureus in E.coli. The active complex is formed by two subunits, GyrA and GyrB. Since S.aureus is a dangerous organism, GyrA and GyrB from S.aureus were cloned into a plasmid. These plasmids can be transformed into E.coli and used as a model for testing the toxicity of diverse substances.

Theoretical Background

For the agar-diffusion assay, bacteria were distributed in liquid LB Agar, which was then poured into petri dishes to solidify. Afterwards, antibiotic wafers were laid on top and the plate was left to incubate. If an antibiotic stops the bacteria from growing or kills the bacteria, there will be an area around the wafer where the bacteria have not grown enough to be visible. This is called a zone of inhibition which depends on the antibiotic activity, the diffusion rate and the solubility of the substance investigated. E.coli are not the perfect chassis for aminocoumarin based antibiotic assays because they have an outer membrane protein, named TolC, which exports aminocoumarins. The lab of Dr. Gust provided us a TolC-defective E.coli strain to circumvent this problem.

Procedure

After transforming TolC-defective E.coli and likewise XL1blue, a colony was picked respectively and used to inoculate 5 mL of LB with selection antibiotics and incubated overnight at 37°C. The next morning 500 µL of the preculture was used to inoculate 5 mL of selection media and grown to an OD600 of ~0.6-0.7. Cells were harvested by centrifugation and resuspended in 100 µL LB without antibiotics. LB-agar was cooked and cooled down until it was hand warm. Bacteria and L-rhamnose were added and the bacteria-containing agar was poured into petri dishes. Wafers (diameter 6 mm, paper, autoclaved) were laid on the solidified LB-agar and 10 µL of an antibiotic stock solution was pipetted onto it. Plates were incubated overnight at 37°C and pictures were taken the following morning.

Results

In order to find a suitable concentration of clorobiocin (small amount, measureable) for our test-system we performed a dilution series of clorobiocin using carbenicillin as a positive control and the respective solvents (MeOH, H2O) as negative control. The radius of the zone of inhibition (z.o.i.) was measured and plotted against the concentration and linear fitted. All measurements were taken as duplicates.

From this experiment we concluded our working concentration of clorobiocin (10µg/µL) for the establishment of the constructs explained in the introduction. iGEM Team Franconia 2017 did exactly the same experiment with Corynebacterium glutamicum, which seems to be very sensitive to clorobiocin.

Our two genes, β-Lactamase and GyrB, are under control of L-rhamnose and L-arabinose dependent promoters respectively. In this way we reduce leaky expression and can start expression at a defined time point. We used Kanamycin as positive control, Carbenicillin was not suitable because the GyrB expression plasmid encodes a Carbenicillin resistance. As negative controls we used the respective solvents and pSB1C3-SHV (BBa_K2372004) and pSB1C3-pRha-RBS (BBa_K2372007) which do not express protein.

Figure 2: TC: Tetracyclin (XL1blue are resistant, 250µg)), Kan: Kanamycin (500µg)

Kanamycin always shows a z.o.i. between 4 and 5.5 mm. XL1blue should be resistant to Tetracycline. The z.o.i. might be a result of the small solubility in water resulting in a steep concentration gradient instead of a more homogenous, thus lower concentration.

Figure 3: Carb: Carbenicillin (1000µg), Kan: Kanamycin (500µg), SHV control: BBa_K2372004, pRha-RBS-SHV: BBa_K2372008. 0.1% (w/v) L-rhamnose⋅H2O was used for induction

The z.o.i. is drastically reduced in XL1blue samples but not that much in the TolC samples. TolC might not be a good expression host.

Figure 4: Clorobiocin (10µg), NDX: nalidixic acid (250µg), Kan: Kanamycin (500µg). 2.5µM of L-arabinose was used for induction.

Nalidixic acid (NDX) is an inhibitor of GyrA, while clorobiocin targets the ATP binding site in GyrB. Therefore NDX can be used as a control. TolC are more sensitive to clorobiocin and NDX than XL1blue. The radius of the z.o.i. in the GyrB expressing samples is reduced in TolC samples. Because the initially used L-rhamnose concentration seemed too low we used different concentrations to find out the most suitable.

Figure 5: 1000µg Carbenicillin in each sample.

The size of the z.o.i. does not decrease directly with the increase in L-rhamnose concentration. TolC seem not expressing protein in response to L-rhamnose.

Discussion

Discussion The measurement of the zone of inhibition in the agar-diffusion assay in E.coli with clorobiocin is challenging because E.coli export aminocoumarins via the outer membrane protein TolC. Furthermore clorobiocin has a low solubility in the water-based LB-agar. The first problem is circumvented by using a TolC knockout variant, the second has to be kept in mind while interpreting the data. Tetracycline has a low solubility in water too and led to small z.o.i. even with the resistant XL1 blue, possibly because of its accumulation and therefore high concentration around the wafer. Very small radii of the z.o.i. (<2mm) as seen in the clorobiocin samples and the discrimination between e.g. 4 and 4.5 mm are hard to measure using a conventional ruler. Sometimes the border of the z.o.i. is diffuse or there is more than one z.o.i. (concentric rings). An automated image analysis system might be better or the determination of “mean inhibitory concentrations” in liquid culture. After all the test-system is working. We were able to make E.coli more resilient against clorobiocin by expressing GyrBR. If we had more time an increasing amount of GyrA and GyrBR from Staphylococcus aureus would have been expressed and validated. The SHV-1 confers resistance to carbenicillin in an inducer concentration dependent manner. The next step would be to design a polycistronic gene structure with a constitutive promoter in order to only need one plasmid without the necessity for external induction.