Team:Tuebingen/Results/Testing
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.
Figure 1: Test results of the clorobiocin dilution series in TolC, XL1blue and C.glutamicum (data from team franconia.
Subsequently, we performed , the actual measurement of 8 different devices as shown in figure 2.
First, plasmids were transformed in DH5-alpha using the standard transformation protocol from iGEM with the deviation of using LB medium instead of SOC medium. For further information on the used protocol go to "http://parts.igem.org/Help:Protocols/Transformation".
Two colonies were picked for each device and incubated in 5-10 mL LB medium + Chloramphenicol (25 µg/mL). The next day the solution was diluted to an OD of 0.02 and 500 µL of the samples were taken and hold on ice at t=0, 2, 4, 6 h. Absorbance (OD600) and fluorescence were then measured using the FLUOstar OPTIMA from BMG LABTECH.
(For detailed protocol click here.)
Figure 2: Workflow InterLab Study 2017
RESULTS AND DISCUSSION
The provided protocol by iGEM was easy to implement by providing a step by step guide to perform the experiments.
Although our data has a high variance between the devices and between the replicates after normalization, device 1 and 2 showed significant higher fluorescence than device 3. This is in line with the data from the device’s reference in the Registry where device 1 was shown to have the highest absorption followed by device 2 and then device 3.
Table 2: Variant RFP with corresponding absorption values
Device 4, 5 and 6 with the Bicistronic Design Element Number 2 showed no real difference in comparison to device 1, 2 and 3 where this element was not present. When the data from all teams is compared we will see if there is a bigger influence on gene expression due to the different promoters used.
At time point 2 h the fluorescence signal was the highest despite for the positive control. If the expression of RFP induces stress, one explanation might be that the bacteria induce expression of proteases or reduce the amount of the necessary transcription factors.
