The Three-Channel Switch
Introduction
We engineered a three-channel switch that can be controlled with the intensity of blue light. By utilizing the mechanisms of lambda (λ) repressor and linking it to a blue light sensor protein, we would be able to swiftly switch between the expressions of three different genes. This mechanism could provide a nearly real-time control over the chosen genes, which could advance a variety of industrial bioprocesses, speed up research projects and benefit metabolic engineering. The switch has a modular structure, and thus, the users can decide the genes themselves without needing to modify the actual mechanism at all.
Features
We chose YF1 fusion protein as the light receptor protein. The receptor autophosphorylates in darkness but in blue light, it is unphosphorylated. A phosphorylated YF1 protein acts as a kinase and activates FixJ transcription factor, which can then bind to a FixK2 binding site and activate the production of λ repressor protein CI. The kinase activity of YF1 is inversely proportional to blue light intensity and the effect is carried on to the acitve FixJ concentration. Thus, CI protein is not produced in bright blue light but the production increases when the blue light is dimmed down.
We used lambda repressor protein CI to regulate the genes in the switch. Only gene A is active when there is no repressor protein CI. At medium concentrations of CI, gene A becomes deactivated and gene B is activated. At high concentrations of CI, only gene C is active. The activity and deactivity of the gene C is based on tetracycline repressor protein that is produced with genes A and B. When neither of those genes are active, gene C is activated. The activities of genes A and B are controlled with an interesting mechanism of lambda repressor, which is explained thoroughly under the background title on this page.
So, to put it all together, in blue light the switch activates gene A, in dim blue light gene B, and in darkness gene C. The image illustrates this functionality.
In blue light the switch activates gene A, in dim blue light gene B, and in darkness gene C. The changes are based on differences in the concentration of the λ repressor protein CI.
Glossary
- YF1 fusion protein = blue-light sensor that becomes unphosphorylated in blue light and phosphorylated (activated) in darkness
- FixJ protein = after being phosphorylated by YF1, this activates promoter FixK2
- FixK2 binding site = activates the production of CI when FixJ binds to it
- lambda (λ) repressor protein CI = a protein that can repress and/or activate the transcription of two different genes
- OR tripartite operator site = an operator site to which CI binds, downstream from OL in λ phage
- OL tripartite operator site = and operator site to which CI binds, upstream from OR in λ phage
- PRM promoter = a promoter that is active only when there’s no CI protein
- PR promoter = a promoter that is active only when there’s little CI protein, too much or too little inhibit the production
- genes A-C = the three genes that you could insert to our system and they would be expressed as explained here
- Tetracycline repressor protein TetR = can bind to TetR repressible promoter sites and inhibit gene transcription
Background
Idea development
/miten alotettiin tutkiminen ja miten nyt mitäkin sit keksittiin/
Earlier research
Uppsala, STJU, Tabor
Light receptor
Lambda (λ) Repressor
Lambda repressor plays a crucial role in our switch. The repressor is part of the lambda phage and it regulates the lysogenic an lytic states of the virus. The lambda phage genetic material can be integrated in the host genome as prophage. This phase is called lysogenic cycle and during that state, the host cell can proliferate and function normally. However, in lytic cycle the phage genetic material is separate from the host genome and it is hevily expressed to produce more viruses which eventually lyse the host cell (hence the name). Lambda repressor is the region in the lambda phage that is responsible for regulating the current state in infected host. This phage has some very interesting characteristics that are described in this subsection.
In the center of the lambda repressor there is a lambda repressor protein CI. This protein represses the genes in the lytic cycle and maintains the lysogenic state. Moreover, this protein is autoregulated in a very special manner. CI dimerizes and binds into its own promoter (PRM) upregulating its own production. However, in high concentrations, CI represses its production maintaining it on an appropriate level. Dimerized CI represses a lytic cycle transcription factor Cro by bindig to operator regions before PR. The lambda phage changes the state from lysogenic cycle to lytic cycle (i.e. lysogenic induction) when the host is exposed to DNA damaging factors, such as UV light. The damage activates the SOS reponse and production of RecA protein involved in repair and maintenance of DNA. RecA has protease activity and it cleaves CI resulting in drop of CI dimer concentration, derepression of Cro and lysogenic induction. Dodd et al (2001) showed that the concentration of CI affects the capability for lysogenic induction. Thus, it is important that lambda phage maintains the CI concentration in sepcific frame in order to be able to switch to lytic cycle when the opportunity arises. High concentration ledads to inability of lysogenic induction whereas low concentration results in too high induction sensitivity.
The interaction of CI protein and lambda repressor promoters is well illustrated in the figure (Dodd et al. 2001). The repressor region consists of two promoters, PRM and PR separated by common operator sites (OR 1, 2 and 3). The CI protein dimers bind DNA in these three operator sites. Johnson et al. (1979) showed that CI binds cooperatively on the three operator sites. The OR1 has the highest affinity for the CI dimer which recruits another dimer to OR2 site. This blocks the transcription of the PR promoter and activates PRM promoter blocking Cro production and activating CI production, respectively. Bell et al. (2000) suggested that these CI dimers at the two operator sites form a tetramer. Very similar interaction is observed between two of the three OL operator sites which are located 2,4kb away from OR operator. It was early that CI also binds to OR3 and OL3 sites, but in substantially higher CI concentrations (Johnson et al., 1979). This leads to repression of PRM promoter. Maurer et al. (1980) showed that 50% repression of PRM requires 15 times the normal lysogenic concentration. However, the mechanism of PRM repression is not result of only CI dimer binding in the OR3 site.
Dodd et al. (2001) showed that two tetramers at OL and OR sites form an octamer by looping the DNA. They proposed that this DNA looping juxtaposes OR3 and OL3 sites and allows them to be linked by a CI tetramer. This, in turn, would silence the PRM promoter. Thus, the lambda repressor has three different states regulated by CI concentration: lytic (low CI concentration, PR is active and PRM is inactive), lysogenic (moderate CI concentration, PR is inactive and PRM is active) and repressing lysogenic (high CI concentration, PR is inactive and PRM is inactive). This is the underlying principle of our three-channel switch. In our approach, we regulate the CI concentration with blue light intensity and replace the Cro and CI with other genes of interest. This way, we are able to use the characteristics of lambda repressor to construct the mechanism behind the three-channel switch.
miten ajateltu muokata omaan systeemiin
The Uppsala 2011 team [2]
SJTU-BioX-Shanghai
Jeff Tabor lab
[Kuva circuitista]
The switch is based on a protein called cI (http://www.uniprot.org/uniprot/P03034). This protein coding sequence is under a promoter that is activated by FixJ, a protein that is phosphorylated and therefore activated by another protein, called YF1. YF1 is phosphorylated and activated in dark. [Lisää!]
We have designed the circuit by using both already existing biobricks and synthesized parts.
We are currently assembling the prototype of our system.
Parts
/parts-lista-igem-template-säätö/
The final construct ended up being fairly large. The gene circuit consists of two different segments: the light sensor that produces CI according to the intensity of light and the actual switch that responds to the differences in the concentration of repressor protein CI.
We used fairly many already existing parts that were in the 2014 iGEM BioBrick Distribution. The parts we created ourselves are /mitä me nyt saadaan lähetettyä/. /selitystä kustakin partista ja että mitä se tekee/.
Here’s a list of all the parts we used in our gene switch. They are in the same order as in our gene circuit and each of them is color coded as follows: /hienot värikoodisysteemit/. /makee luettelo niistä brickeistä/.
This is the complete sequence that we put together. The color codes are the same as in the previous list. Gene A has 20 Xs as placeholders, gene B has 20 Xs and gene C has 20 Xs. As anyone could decide the genes themselves, the placeholders are just to show the correct place in the sequence.
In addition to these parts, we also used a GFP part for testing the response times of YF1. /ja mitä muuta nyt ollaankaan testailtu/
/genecircuitkuva/ https://static.igem.org/mediawiki/2014/f/f4/Aalto_Helsinki_Gene_Circuit.png
[kuvateksti] This is our gene circuit. The upper part is the light sensing segment that produces the CI protein and the lower part reacts to differences in the CI concentration and switches the gene channel. The turquoise arrows are promoters, the turquoise circles are operator sites, the light blue circles are ribosome binding sites, the gray squares are expressed genes. The promoter of YF1 gene can be any constitutive promoter.
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