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
• O_R tripartite operator site = an operator site to which CI binds, downstream from O_L in λ phage
• O_L tripartite operator site = and operator site to which CI binds, upstream from O_R in λ phage
• P_RM promoter = a promoter that is active only when there’s no CI protein
• P_R 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

In the beginning of our project, from March 2013, we focused solely on coming up with new ideas, building the public team website and applying for Summer of Startups 2014 and foundations with deadlines in spring. In our team meetings, we had different types of brainstorming sessions where all of us could presenet even the craziest ideas. We found a shared folder in the cloud where anybody could write up new ideas. We came up with disco bacteria that glow in sync with music, new kinds of bacterial drinks that change flavour with storing temperature, remote controllable bacteria, oil-eating bacteria, tooth paste bacteria that keeps up the mouth hygiene despite forgetting to brush the teeth, etc. On evening we had a vote and everybody gave a score from 1 to 5 for each idea. We made an idea gradient where the ideas were ranked according to the score from bottom left to top right.

We chose the top 5 ideas and presented them to our advisors and researchers from Aalto University, VTT and University of Helsinki to get feedback and comment on the feasibility. After all, none of us had never done anything even close to this before. As a result of this feedback, further idea development and admission to Summer of Startups, we decided to work on the idea with a project name "nest box." It's a container resembling a traditional Finnish nest box but it includes bacteria producing different fragrances. These bacteria could be controlled using light concealed inside the box. The user can decide which fragrance to produce by a press of a button and this action would be translated into light signal to control the gene expression in the bacteria. Such a system is concrete and understandable for the public audience and it would help to build a positive image of synthetic biology in Finland. In theory, such system could be used in conference rooms, hotels, living rooms, saunas, movie theaters... The scent marketing is an expanding business and our idea had the potential to add something new to it. Furthermore, it was a concrete product which made our participation in Summer of Startups a lot easier.

We started our practice project mid May and Summer of Startups early June. As we got forward, we realized that our idea is not very realistic. Implementation of such a separate unmaintained container would be very challenging and in addition, we cannot bring genetically modified bacteria outside laboratory conditions (it's defined in the gene technology legislation). We decided to solely focus on one element of our nest box idea. Constructing the interface between the bacteria and the user or a computer is not trivial. So, we redefined our direction which was maintained to the end. We wanted to develop a three channel switch that would allow us to activate one of the three genes at a time using only the intensity of single wavelength light. We could then connect this switch with fragrance producing genes, for example.

Earlier research

Uppsala, STJU, Tabor

Light sensing system

First of all, to be able to control gene expression with light, we needed a light receptor protein. It would react to certain wavelength of light and mediate the signal to the DNA level and initiate the response. We found different alternatives in the iGEM registry but most of them were not properly characterized. We did not found reliable experiences of the use of such light receptors or when we found them, the receptor protein was not available. Our requirements for the light receptro system were following: 1) it has worked for other iGEM teams 2) it should work in E. coli 3) it is available in the registry 4) it does not require any cofactors or special strains 5) short response time and sensitivity. In this section, we discuss about the different alternatives we found and what made us choose the YF1 protein. We also introduce the mechanism how the light signal is conveyed in our system.

Phytochrome interacting factors (PIF's) were used by some previous iGEM teams, including Freiburg 2013 and TU Munich 2012. The actual red light sensing protein is called Phytochrome B and in the active form it binds to the PIF. Thus, the cell also needs to express Phytochrome B. However, these PIFs were derived from Arabidopsis thaliana, which is an eukaryotic organism. Moreover, the response mechanism would require fusing the two proteins to the actual DNA binding domain and transcription factor. Unfortunately, Freiburg team only succeeded in 1.3 fold induction of gene expression after 48 hours of incubation in red light and TU Munich did not manage to implement the light sensing part in their kill switch.

The Peking 2012 team designed an ultra-sensitive light sensor for luminescence. They combined a LOV domain, a dimerizing domain and a DNA-binding LexA domain. Such protein becomes active when the cells are illuminated with 440 - 480 nm light. In active form, the dimerizing domains facilitate formation of LexA dimer which is able to bind DNA and repress transcription in only such form. Whereas their light sensing system seems to work very well there were three issues with it. Firstly, their light sensing system was not available and secondly, it might have been too sensitive; controlling the light at very low intensities could be rather challenging. Furthermore, LexA is a part of SOS system already part of E. coli so it might interfere with indigenous E. coli genes. However, according to Peking 2012, mutated version of LexA could solve this problem.

Cph8 is a fusion of Cph1 red photoreceptor domain (660nm) and EnvZ histidine kinase domain. In absence of red light, EnvZ is active and it phosphorylates endogenous OmpR which in trun activates OmpC promoter. Red light activates Cph1 which inhbits EnvZ kinase domain function. However, as both EnvZ, OmpR and OmpC are endogenous in E. coli, reliable use would require EnvZ deficient strain. The activation Cph1 requires Phycocyanobilin which can be produced with heme oxygenase and ferrodoxin oxidoreductase. The Cph8 was decribed by Levskaya et al. (2005) and they achieved nearly 10-fold induction. However, as EnvZ and OmpR are involved in indigenous osmolarity response, phosphorylated OmpR might also interfere with promoters encoding membrane porins.

CcaS is a cyanobacteriochrome that gets activated by autophosphorylation. This is upregulated by green light (535nm). The activated CcaS phosphorylates its response regulator CCaR which then induces the promoter P_cpcG2 (Hirose et al., 2008; Tabor et al., 2011). As Cph8 system, this light receptor also requires Phycocyanobilin to function. In addition, CcaS is inactivated in red light (672nm) allowing higher degree of control and fast response times. According to the experiments by Tabor et al. (2011), the promoter P_cpcG2 showed only 2-fold induction in green light. The transcription response is somewhat linear to the light intensity of 0.01 W/m² after which is rapidly saturates. Although CcaR and CcaS BioBricks are indeed available although the experiences of other iGEM teams were very few and poorly described.

YF1 is an engineered blue light (previous experiments in 430nm) sensor protein that consists of two initially separate domains, LOV domain (sensory domain) from YtvA and histidine kinase domain (effector domain) from FixL. This fusionprotein was designed and constructed by Möglich et al. (2009). YF1, nor it's precursors, do not exist naturally in E. coli nor reportedly interfere with indigenous E. coli genes but according to Möglich et al. (2009) the sensor works well in E. coli: it's capable of 70-fold supression of the target promoter when illuminated with blue light. The sensor does not require additional, foreign molecules in order to get activated by blue light. The gene is only 1132 bp long and it has been used by previous iGEM teams, including Uppsala 2011, STJU BioX Shanghai and Tokyo NoKoGen. Thus, we evaluated YF1 to be the most suitable for our three channel switch.

Based on the work of Möglich et al. (2009) we describe the mechanism of YF1 blue light sensor in greater detail. YF1 is a water soluble chimeric fusionprotein that contains two domains linked together with a Jα linker sequence. The N terminal domain is a flavin nucleotide binding LOV domain (light-oxygen-voltage, a sub group of PAS sensor domains) from Bacillus subtilis YtvA. This domain incorporates noncovalently flavin mononucleotide and blue light facilitates formation of a covalent bond with a cysteine residue of the LOV domain. This induces a conformational change in this light sensory domain.

The C terminal domain of YF1 is a histidine kinase, the FixL effector domain from Bradyrhizobium japonicum. The original protein, FixL, is involved in regulating the nitrate respiration and nitrate fixation. The histidine kinase functions only when it is phosphorylated. It has autophosphorylation activity that is regulated by the sensor domain. Autophosphorylated histidine kinase activates a FixJ response regulator by transferring a phosphoryl group. However, when the histidine kinase domain is unphosphorylated, it has actually phosphatase activity for the response regulator. Phosphorylated FixJ binds to FixK2 promoter initiating the expression of the corresponding genes. This system should be completely orthogonal in E. coli.

Möglich et al. (2009) proposed that the YF1 proteins form dimers. This would also bring the histdine kinase domains close to each other. It is proposed that the autophosphorylation actually work in trans within the dimer. They hypothesized the Jα helix linker to form a coiled coil with another YF1 protein. The linker would mediate the signal from the sensor domain to the effector domain inhibiting/enabling its kinase activity. The conformational change from the incorporated flavin domain is suggested to rotate the linker helix. The relative angle of the two domains greatly affect to the mechanism. If we add one amino acid to the Jα linker, the relative angle changes and the effect of the sensor domain is inverted: in presence of light the histidine kinase is active but in darkness, it is inactive. This presents interesting approaches and easy solution to invert the YF1 signal.

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 (P_RM) 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 P_R. 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, P_RM and P_R separated by common operator sites (O_R 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 O_R1 has the highest affinity for the CI dimer which recruits another dimer to O_R2 site. This blocks the transcription of the P_R promoter and activates P_RM 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 O_L operator sites which are located 2,4kb away from O_R operator. It was early that CI also binds to O_R3 and O_L3 sites, but in substantially higher CI concentrations (Johnson et al., 1979). This leads to repression of P_RM promoter. Maurer et al. (1980) showed that 50% repression of P_RM requires 15 times the normal lysogenic concentration. However, the mechanism of P_RM repression is not result of only CI dimer binding in the O_R3 site.

Dodd et al. (2001) showed that two tetramers at O_L and O_R sites form an octamer by looping the DNA. They proposed that this DNA looping juxtaposes O_R3 and O_L3 sites and allows them to be linked by a CI tetramer. This, in turn, would silence the P_RM promoter. Thus, the lambda repressor has three different states regulated by CI concentration: lytic (low CI concentration, P_R is active and P_RM is inactive), lysogenic (moderate CI concentration, P_R is inactive and P_RM is active) and repressing lysogenic (high CI concentration, P_R is inactive and P_RM 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.

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.

Hypothesis

Light-to-CI, BioBrick convenience, OR=OL

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/

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.

Our Research Methods

LED-rig for excitation

To be able to shine light on our bacteria we designed a new kind of device from scratch. Inspired by the rig developed by Tabor labs¹ we decided to build a simpler one, only with blue LED:s.

Luckily we have some electronic knowledge in our team too. With Pietu designing the rig we built a foam-padded transportable rig that can be put in an incubator overnight.

Microcontroller

LED:s

Constructing

1. E.J. Olson, L.A. Hartsough, B.P. Landry, R. Shroff, J.J. Tabor,
   "Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals."
   Nature Methods 11(4), 449-455 (2014).
2. Arduino Nano microcontroller, http://arduino.cc/en/Main/arduinoBoardNano
3. Adafruit 16-channel PWM shield, https://learn.adafruit.com/adafruit-16-channel-pwm-slash-servo-shield?view=all
4. S. Muthu, F. Schuurmans and M. Pashley, “Red, Green and Blue LED based white light generation: Issues and Control,” 37th Annual IEEEIAS meeting, Vol. 1, pp. 327 – 333 (2002)
5. http://www.thermoscientific.com/content/tfs/en/product/varioskan-flash-multimode-reader.html

Results

Discussion

Mutaatiot operaattorialueisiin Plasmidissa ei DNA:ssa Solutason kohina solusyklissä

Conclusions

References

1. Peking iGEM Team (2012), https://2012.igem.org/Team:Peking
2. Uppsala-Sweden iGEM Team (2011), https://2011.igem.org/Team:Uppsala-Sweden
3. SJTU BioX Shanghai iGEM Team, (2013), https://2013.igem.org/Team:SJTU-BioX-Shanghai
4. Dodd, I. B., Perkins, A. J., Tsemitsidis, D., & Egan, J. B. (2001). OcDodd, I. B., Perkins, A. J., Tsemitsidis, D., & Egan, J. B. (2001). Octamerization of lambda CI repressor is needed for effective repression of P(RM) and efficient switching from lysogeny. Genes & Development, 15(22), 3013–22. doi:10.1101/gad.937301tame. Genes & Development, 15(22), 3013–22. doi:10.1101/gad.937301
5. Hirose, Y., Shimada, T., Narikawa, R., Katayama, M., & Ikeuchi, M. (2008). Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proceedings of the National Academy of Sciences of the United States of America, 105(28), 9528–33. doi:10.1073/pnas.0801826105
6. Johnson, A. D., Meyer, B. J., & Ptashne, M. (1979). Interactions between DNA-bound repressors govern regulation by the lambda phage repressor. Proceedings of the National Academy of Sciences of the United States of America, 76(10), 5061–5. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=413079&tool=pmcentrez&rendertype=abstract
7. Johnson, A. D., Poteete, A. R., Lauer, G., Sauer, R. T., Ackers, G. K., & Ptashne, M. (1981). λ Repressor and cro—components of an efficient molecular switch. Nature, 294(5838), 217–223. doi:10.1038/294217a0
8. Levskaya, A., Chevalier, A. A., Tabor, J. J., Simpson, Z. B., Lavery, L. A., Levy, M., … Voigt, C. A. (2005). Synthetic biology: engineering Escherichia coli to see light. Nature, 438(7067), 441–2. doi:10.1038/nature04405
9. Möglich, A., Ayers, R. A., & Moffat, K. (2009). Design and signaling mechanism of light-regulated histidine kinases. Journal of Molecular Biology, 385(5), 1433–44. doi:10.1016/j.jmb.2008.12.017
10. Tabor, J. J., Levskaya, A., & Voigt, C. A. (2011). Multichromatic control of gene expression in Escherichia coli. Journal of Molecular Biology, 405(2), 315–24. doi:10.1016/j.jmb.2010.10.038
11. Zopf, C. J., Quinn, K., Zeidman, J., & Maheshri, N. (2013). Cell-cycle dependence of transcription dominates noise in gene expression. PLoS Computational Biology, 9(7), e1003161. doi:10.1371/journal.pcbi.1003161
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