Design

In organisms that are resistant to tetracycline, the resistance genes are not always transcribed. The genes only become transcribed in the presence of tetracyclines in order to save energy and conserve resources for the cell. Figure 1 shows the basics of this gene repression and activation system which forms the basis for our biosensor. By replacing the resistance genes downstream of the operator and promoter, we could introduce a reporter gene that will become active in the presence of tetracycline. Our goal is to have an Escherichia coli cell that is able to sense the presence of tetracycline in a sample and produce a colour change to notify the user of a positive result. To avoid the risk of having all tetracycline pumped out of the cell before the system can be activated, we will try to not use the tetracycline efflux pump to confer resistance to our E. coli cells. This will require us to test the level of resistance innately found in the E. coli strains.

Figure 1: Overview of the tetracycline operon repression and activation system. TetR binds to the tetO sequence repressing the transcription of downstream genes. Tetracycline can bind to TetR causing a confirmation change resulting in TetR losing its ability to bind to tetO. This leaves the operator and promoter sequence open to RNA polymerase binding and the transcription of downstream genes can begin.

To start, we will use the plasmid named pJKR-H-TetR (hereby referred to as pTet) as a backbone. Currently this plasmid has GFP downstream of the Tet operator (Figure 2) as the reporter. We plan to transform the common E. coli lab strains BL21(DE3) and DH5a with this plasmid. The problem with using this plasmid with GFP is that fluorescence is needed in order to take measurements which is not useful to applications outside of the lab.

Figure 2: Basic schematic of pTet tetracycline reporter system using GFP.

To make the system able to detect tetracycline in a manner that provides visual feedback to a user, the blue chromoprotein amilCP (BBa_K592009) was used and put downstream from a tetracycline inducible promoter (BBa_K3189001) (Figure 3). The amilCP chromoprotein was originally from the coral Acropora millepora and was submitted by iGEM Uppsala in 2011². When tetracycline is added to this system, a blue colour should be produced. This original amilCP gene has been used and characterized by many iGEM teams and it has been established that amilCP exerts a fitness cost on the bacterial organisms that produce it³. This is why we will also be using an improved version of amilCP created by iGEM Uppsala 2018 (BBa_K1349002) that should be more stable than BBa_K592009.

Figure 3: Basic schematic of the proposed amilCP tetracycline reporter system. In this figure, tetO corresponds with BBa_K3189001 and amilCP corresponds with either BBa_K592009 resulting in the new composite part BBa_K3189015 or BBa_K1349002 resulting in the new composite part BBa_K3189014. Neither new composite part contains the tetR (BBa_K3189004) sequence.

We do see a problem with using amilCP as the reporter in the final biosensor. If the sample tested has no tetracycline there will be no colour change as expected, but in the event that there is so much tetracycline that our E. coli cells die there will also be no colour change. This leaves the potential for false negatives which would not result in an effective and reliable biosensor.

To combat the problem of false negatives, we proposed using an alternate reporter system. We think that the violacein biosynthesis pathway would work for our application as its branching nature (Figure 4) will allow for a built-in negative control. This pathway was isolated from Chromobacterium violaceum and results in the production of the pigment violacein⁴. We plan on using only the VioA (BBa_K3189010), VioB (BBa_K3189011), VioC (BBa_K3189000; violacein synthase⁵), and VioE (BBa_K3189012) enzymes which will produce deoxyviolacein (pink) and prodeoxyviolacein (green). The VioD enzyme, which is needed for full violacein production, will be omitted from this construct to allow for future expandability of this system.

Figure 4: Violacein biosynthesis pathway. The VioABE enzymes convert L-tryptophan into the first intermediate which will spontaneously decompose into prodeoxyviolacein. If VioC is present, the intermediate is transformed into deoxyviolacein. VioD allows for the production of another intermediate which can result in two other colours and has potential for future expansion of the systems sensing capability. This figure was adapted from iGEM Washington 2017.

Our plan is to put vioABE under the control of a modified lac promoter (BBa_K3189013) to only become active in the presence of lactose or the lactose analogue IPTG, which we plan on providing our system⁴. Next vioC will be under the control of the tet operator (BBa_K3189005) to become active only in the presence of tetracycline. An overview of these constructs is provided in Figure 5and a chart of gene activation is provided in Figure 6. When implemented in our bacterial system it should behave as shown in Figure 7 with a positive sample turning the system pink and a negative sample will turn the system green. The advantage of this system over the previous pigment systems is the fact that a non functioning system will alert the user by producing no colour change.

Figure 5: Basic schematic of the implementation of the violacein biosynthesis pathway as the reporting system for tetracycline presence. The tetO (BBa_K3189001), vioC (BBa_K3189000), and tetR (BBa_K3189004) construct (known all together as BBa_K3189005) will exist at a different point in the genes than the vioABE (BBa_K3189013) composite part when transformed into the E. coli cells.

Figure 6: Enzyme and pigment production predicted for tetracycline and IPTG presence or absence. The enzymes crossed out in each conditions indicate they are not being produced under those conditions. The background of the squares indicates the pigment colour expected under those conditions.

Figure 7: Predicted function of the biosensor in E. coli cells. When no tetracycline is present the cells will produce a green pigment. When there is tetracycline, the cells will produce a pink pigment.

References

1. Rogers, J. K. et al. Synthetic biosensors for precise gene control and real-time monitoring of metabolites. Nucleic Acids Res. 43, 7648–7660 (2015).
2. Levy, O. et al. Light-responsive cryptochromes from a simple multicellular animal, the coral Acropora millepora. Science 318, 467–470 (2007).
3. Liljeruhm, J. et al. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. J. Biol. Eng. 12, 8 (2018).
4. Cress, B. F. et al. Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli. Nucleic Acids Res. 44, 4472–4485 (2016).
5. Brazilian National Genome Project Consortium. The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc. Natl. Acad. Sci. U. S. A. 100, 11660–11665 (2003).

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