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

Parts

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.

Background

How the Research Began

/miten alotettiin tutkiminen ja miten nyt mitäkin sit keksittiin/

Lambda (λ) Repressor

/lambda-repressor toiminta, eli miten CI bindaa ja mitäkin repressoituu, looppaus/ O_R-, O_L-vertailu 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.

References

1. Peking 2012 iGEM Team: https://2012.igem.org/Team:Peking
2. Uppsala-Sweden 2011 iGEM Team: https://2011.igem.org/Team:Uppsala-Sweden
3. SJTU BioX Shanghai 2013 iGEM Team: https://2013.igem.org/Team:SJTU-BioX-Shanghai

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, onli 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

References