Inspiration

Nothing in pipeline? - The problem of modern antibiotic research

Worldwide globalisation and a rising world population creates a new problem for our society: Pathogens acquire mutations and different resistance factors leaving us with less and less effective antimicrobials. The World Health Organisation (WHO) considers the most problematic organisms to be multi-resistant Mycobacterium tuberculosis, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and many others (Geneva: World Health Organization 2017).

Besides fighting antibiotic misuse by raising awareness and health education to decrease the number of new resistant bacteria, there is a necessity for new antibacterial substances and therapies. This lies within the responsibility of universities and companies. In contrast, every year, the FDA approves less and less new antibacterial compounds. This is due to the fact, that research in the field of new antibiotics for pharmaceutical companies is not attractive: New substances are usually only used as reserve antibiotics for patients suffering from infections with highly resistant pathogens and therefore provides in its patented period only a low budget income.

Most new antibiotic substances are found in new bacterial strains, while (bio-)chemically modified compounds are rather rare. At the same time, it gets harder to identify new potent candidates. We need new strategies to produce effective compounds that are specifically engineered to fight resistant pathogens and new methods to achieve these modifications. This is where antibiotic classes, that provide potency in theory, but fail in compliance and practical application, come into play.

Introducing the aminocoumarins: potent antibiotics with little relevance in clinical therapy

Aminocoumarins are one of many substance classes with almost no use in the antibacterial therapy. So far, three aminocoumarins are known: Novobiocin, Clorobiocin and Coumermycin A1. (Heide 2014) Furthermore, the structurally different Simocyclinone D8 has been described as a fourth aminocoumarin (Schimana et al. 2000).

All test devices are composite parts containing GFP with constitutive promoters, a negative control without GFP is also included. The vector pSB1C3 has a chloramphenicol resistance. Device 4, 5, and 6 additionally contain a Bicistronic Design Element Number 2 which was designed by Mutalik et al. in a2013 Nature publication. This element should induce precise and reliable gene expression.

Mutalik, V. K. et al. "Precise and reliable gene expression via standard transcription and translation initiation elements." Nature Methods 10, 354–360 (2013).

For normalization standard curves were made with the provided measurement kit from iGEM.

PRACTICAL WORKFLOW

Before the actual measurement, calibration was performed for OD600 and a fluorescence standard curve was determined using a clear bottom black 96-well plate in four replicates.

Figure 1: Fluorescein standard curve obtained by dilution series of fluorescein in 4 replicates.

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.

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.

Figure 3: Results show in µM Fluorescein/OD600 for Devices 1, 2, 3 in comparison to devices 4, 5, 6. Samples were taken at t = 0, 2, 4, 6 h. Values smaller than 0 were excluded in the graphic. Biological duplicates are represented from each device. BCD2: Bicistronic Design Element Number 2.