Team:UANL Mty-Mexico/Project/Mechanism

Light sensing proteins have been successfully characterized in the last few years[1]. Dr. Jeff Tabor (2010) have created a two-photoreceptor system in E. coli and described the specifics required for them to work both separately and together (figure 1). Two light sensors will be used in this project, responsive to green and red light respectively.

Figure 1. Schematic representation of the engineered two-color light induction system in E. coli as constructed Dr. Jeff Tabor. Figure taken from Tabor JJ et al. (2010) J Mol Biol 405:315-324.

Both sensors share a common chromophore and light absorption mechanism but have different chromatic specificities and transcriptional outputs. The green sensor has two genes, CcaR and CcaS. Light exposure increases the rate of CcaS autophosphorylation, phosphotransfer to CcaR, and transcription from the promoter of the phycobilisome linker protein cpcG2. The red light-sensing protein Cph8 is expressed in the phosphorylated ground state. It is switched to the unphosphorylated state by 650 nm light and back to the phosphorylated state by 705 nm light. When phosphorylated, Cph8 passes a phosphoryl group to OmpR, which then binds to and activates transcription from P_ompC. Because it is inactivated by red light, Cph8 can be considered a logical (NOT red) sensor. A genetic inverter or logical NOT gate is used to invert the response of the (NOT red) sensor to that of a red light sensor[1].

Genome integration of large constructions in E. coli 's genome has been proven successful as well[2]. As the use light induction at iGEM competitions and synthetic biology is increasing, we decided to create a built-in light induction system in E. coli.

We intend to integrate the necessary genetic cassette into an appropriated E. coli strain in order to make it capable of light-induced gene expression through two lights -red and green lights- without the need of any extra-chromosomal DNA. We decided to use the method designed by Kuhlman et al. (2010) because it is suitable for the insertion of large DNA fragments into any desired location in the E. coli chromosome. We believe this new chassis could become a useful tool in the light-induction field.

The dual response that depends on the duration of the pulse in cells one and two relies on a so-called biphasic switch. Biphasic switches combine positive and negative regulation so that they are only turned on by a small band of input. A promoter can be made biphasic by introducing a binding site where a regulator can behave as an activator and one where it behaves like a repressor. When the regulator has a higher affinity for the first site, then small concentrations induce transcription and larger concentrations repress it[3].

Figure 2. Behavior of wild type (wt) Lambda's pRM. LacZ gene is placed under pRM's control. At low concentrations of cI, lacZ expression is stimulated. At high concentrations of cI, lacZ gets repressed. Figure taken and modified from Dodd BI et al (2001) Gene Dev. 15:3013–3022.

As cI promoter from phage λ behaves as a natural biphasic switch (figure 2)[4][5][6], we decided to borrow the mechanism. The biphasic switch allows controlling both expression and repression of a promoter by varying the intensity of a single stimulus. Our genetic circuit takes advantage of that to control the independent expression of two different promoters using that single stimulus. It does it through an embedded biphasic switch in a rather complex toggle switch. A toggle switch usually uses two repressors that cross-regulate each other's promoter. A gene of interest can be placed under the control of the same promoter as the repressor so it makes part of the switch. As cI protein can activate and repress, we figured that it could be used to:

1) Control the expression and the repression of pRM promoter (remember, it is a biphasic switch). Placed under pRM will be cI434, a repressor which acts on pRM434 (a different promoter).

2) Control the expression of pRM434 which can be activated by cI and repressed by cI434. This promoter has nothing to do with the biphasic switch though, it merely allows expression when binding of cI and repression when cI434 bound to its operator.

The expected behavior will be that at low concentrations of cI, both promoters will be activated. However, because of the cI434 repressing pRM434, only pRM will stay on eventually. In sake of clarity, from now on we will assume low concentrations of cI will only turn on pRM. At high concentrations of cI the first promoter -and consequently cI434- will be turned off, allowing pRM434 to be activated by cI.

Once explained a general overview of the biphasic switch, we are able to connect the dots with our light-code. cI will be placed under the control of an inducible promoter, which will be activated by light. A pulse of light will activate cI's expression for a short time and result in low cI concentrations, which will only activate pRM promoter (because of cI434 repressing the second promoter). If a continuous light is applied to the cell, cI concentration will increase. As it increases, the repression threshold of pRM will be reached, eventually leading to the activation of pRM434 (as cI434's stops being produced and the remaining degrades). Now we can place any gene under the control of pRM -next to cI434- and any gene under the control of pRM434, managing to control the independent expression of both products through one single light. In this case, different reporter genes are placed under each promoters' control. That is how cells one and two will differentiate between continuous and pulsatile light signals. The concept is simple, pulsatile light leads to low [cI], while constant light leads to high [cI].

The next graph illustrates the expected behavior for cell one, which has GFP and YFP as reporter genes. The behavior would be the same for cell two, with its respective reporter genes.

Figure 3. Expected behavior of cell one. GFP and cI434 are placed under the control of pRM. At low concentrations of cI, GFP's and cI434's expression are stimulated. At high concentrations of cI, GFP and cI434 get repressed, allowing the expression of YFP. Low [cI] would be achieved through pulsatile red light, while high [cI] will be achieved through constant red light. The data used for this graph is not real, for explanation purposes only.

Lambda cI protein has the capability to bind several operators within a complex regulatory region that involves mainly three promoters (pRM, pR and pL). There are two well-known sets of cI binding sites in lambda (OR and OL) spaced 2.4 kb apart and composed of three operators each (OR1:OR2:OR3; OL1:OL2:OL3) (figure 4)[7].

Figure 4. The λ operators OL and OR. The promoter pRM transcribes cI, rexA, and rexB in a lysogen, which sum up to size of ∼2.4 Kb. Figure taken and modified from Court DL et al. (2007) J Bacteriol 189:298–304.

cI has a higher affinity for OR1 and OR2 operators, where binding positively regulates the pRM promoter. On the other hand, when bound to OR3 cI represses the promoter; however, due to a low binding affinity, a higher concentration of the protein is required. Thus, cI can act as both an activator -at low concentrations- and a repressor -at high concentrations- on pRM.

Nonetheless, it has been proven that cI's binding affinity to OR3 is not strong enough to keep pRM repressed[4]. But, in presence of the OL set, the protein can octamerise specifically binding to the two sets of operators at the same time and therefore forming a DNA loop (figure 5)[8]. Such structure stabilizes cI's binding to OR3, allowing pRM's repression at high concentrations.

Figure 5. (a) EM picture of a circular relaxed plasmid with a large DNA loop formed by cI. Two tetramers on each DNA circle interact to form an octamer and thereby loop the DNA into a structure resembling a figure of eight. (b) Close up cartoon representing cI octamers occupying the OL1, OL2, OR1, and OR2 operators. OR3 is to be occupied as [cI] increases. The lower portion shows the sequence of PRM and OR3, the latter between the -10 and -35 where cI cooperative binding inhibits transcription. Figures taken and modified from: Révet, B. et al. (1999) Curr Biol 9:151–154 and Dodd IB et al (2001) Genes Dev 15:3013–3022, respectively.

The third cell has a more simple mechanism, probably a familiar one. As you may figured it out, cell three uses a logic gate called AND gate. That means it requires two inputs to be on (figure 6), red and green lights for this case.

Figure 6. AND gate's Truth table. Output results only if input A and B are present at the same time.

For this project, we designed an AND-gate based on repression (which could be considered as a NOR gate, but we will not make it more complicated). A repressor's gene is placed under the control of two different promoters. Each promoter is as well repressed by one light (through a different intermediate repressor), so that only when both lights are on the reporter gene can be expressed (figure 7).

Figure 7. Repression based AND-gate. A repressor's gene, which acts on the reporter gene, is placed under the control of two different promoters. Each promoter is as well repressed by one light (through a different intermediate repressor), so that only when both lights are on the reporter gene can be expressed.

1. Tabor JJ, Levskaya A, Voigt CA (2010) Multichromatic Control of Gene Expression in Escherichia coli. J Mol Biol 405:315-324.
2. Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs. Nucleic Acids Res 38:e92.
3. Voigt AC (2006) Genetic parts to program bacteria. Curr Opin Biotechnol 17:548–557.
4. Dodd BI, Perkins AJ, Tsemitsidis D, Egan BJ (2001) Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Gene Dev 15:3013–3022.
5. Isaacs FJ, Hasty J, Cantor CR, Collins JR (2003) Prediction and measurement of an autoregulatory genetic module. Proc Natl Acad Sci USA 100:7714-7719.
6. Michalowski CB, Short MD, Little JW (2004) Sequence tolerance of the phage λ PRM promoter: implications for evolution of gene regulatory circuitry. J Bacteriol 186:7899-7999.
7. Court DL, Oppenheim AB, Adhya LS (2007) A New Look at Bacteriophage λ Genetic Networks. J Bacteriol 189:298–304.
8. Révet B, von Wilcken-Bergmann B, Bessert H, Barker A, Müller-Hill B (1999) Four dimers of repressor bound to two suitably spaced pairs of operators form octamers and DNA loops over large distances. Curr Biol 9:151–154.