Team:Aalto-Helsinki/Research

From 2014.igem.org

(Difference between revisions)
Line 117: Line 117:
   <h2>Parts</h2>
   <h2>Parts</h2>
-
   <!--</html><groupparts>iGEM014 Aalto-Helsinki</groupparts><html> -->
+
   </html><groupparts>iGEM014 Aalto-Helsinki</groupparts><html>
-
 
+
  
-
   <p>JUST FOR TESTING</p>
+
-
  </html><groupparts>iGEM013 NCTU_Formosa</groupparts><html>
+
   <p>
   <p>
   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.
   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.

Revision as of 14:18, 30 September 2014

Research

We engineered a three-channel switch that allows you to choose which of its three genes is active. The switch is designed so that the user can define the expressed genes independently.

Scroll down to read more
The Switch
Parts
Background
Methods
Results
Discussion
References

Photo © Tanja Maria

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

Parts

<groupparts>iGEM014 Aalto-Helsinki</groupparts>

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.

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/ OR-, OL-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

The core of the rig is an Arduino Nano2 microcontroller. The Arduino is responsible for the logic behind our illumination patterns. The patterns in our experiments are fairly simple and could in theory be done without the Arduino, but we chose to use it due to its wide popularity and because we now could make advanced (and cool) animations on our rig. Also the Arduino acts as a 5v voltage regulator which provides the voltage for the second part of the rig: the Adafruit 16-channel 16-bit PWM controller3.

LED:s

Light Emitting Diodes(LEDs) are current controlled components, and their intensity depends on the current passing trought them. In our project we used miniature leds because of their small power and small heat generation. It is important that the LED:s wont heat up the bacteria remarkably. Controlling the intensity of these leds is trivial by varying the current, but this will cause the emission spectra to shift3. Hence we choose a different, and very popular, method for varying LED intensity: Pulse Width Modulation (PWM). The idea behind PWM is to turn the led on and off very fast, up to frequencies of 1.6kHz. By varying the time the led is on and off we will be able to make a percieved difference in intensity. The bigger the ratio of on-time versus off-time the brighter the LED.

Due to the hurry of making this rig we chose to use a pre-made Arduino "shield" for this task. We chose the a 16-channel 16-bit PWM shield from Adafruit. The 16-bits provide us much more range than the standard 8-bits of the Arduino. Additionally the Nano does not support as many channels so having 16 LEDs provides more flexibility in our measurements.

Constructing

We wanted to be able to illuminante a bacteria culture with a single led. We choose to culture our bacteria on microtiter-plates. This choice was also affected by the microplate reader we used to measure fluorescence (the GFP). We had the chance to get to use a Varioskan5.
We designed our system so that a unmodified 96 well microtiter plate could be inerted easily. Our LEDs are attached to a lid that can be placed on the microtiter plate. 16 LEDs will illuminate the wells on columns 11,9,7,5 and rows B, D, F, H.

The LEDs we had a peak value of 470nm. This value is specified by the manufacturer, but the spectra of individual leds can vary considerably. Therefore we will measure the spectra od our LED:s with a spectrophotometer to be sure of the excitation wavelength.
We have yet to invent a way to measure the LED intensity. If you have ideas, pleaser contact us!

  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

fl

Results

Discussion

References

Retrieved from "http://2014.igem.org/Team:Aalto-Helsinki/Research"