Team:MIT tconst

From 2010.igem.org

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<div class="bodybaby" id="improving">Improving R0065</div><br>
<div class="bodybaby" id="improving">Improving R0065</div><br>
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R0065 is a hybrid promoter that the MIT 2010 iGEM team used in our composite parts to control transcription of mCherry fluorescence. In conjunction with our toggle, pLPTa, this promoter would only allow expression of fluorescence in the presence of AHL, LuxR, and UV exposure. While we found the promoter very useful, it is described many times on its <a href="http://partsregistry.org/Part:BBa_R0065">parts registry page</a> as being "leaky". After looking into the promoter, we realized this was due to two -10 promoter sequences, possibly due to an error when designing the hybrid promoter.
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R0065 is a hybrid promoter that the MIT 2010 iGEM team used in our composite parts to control transcription of mCherry fluorescence. In conjunction with our toggle, pLPTa, this promoter would only allow expression of fluorescence in the presence of AHL, LuxR, and UV exposure. While we found the promoter very useful, it is described many times on its <a href="http://partsregistry.org/Part:BBa_R0065">parts registry page</a> as being "leaky". After looking into the promoter, we realized this was due to two -10 promoter sequences, possibly due to an error when designing the hybrid promoter.<br><center><img src="http://2010.igem.org/wiki/images/a/af/Leakiness.png"><br>In this FACS scatter, the x-axis represents green fluorescence, and the y-axis represents red fluorescence with zero UV exposure. Theoretically, there should be no red fluorescence. The red fluorescence in part K415023 is controlled by the original R0065, and in K415069 is controlled by our less leaky promoter K415031. You can see that the original promoter is more leaky than our fixed promoter: 3% of K415069 fluoresce red, while 15% of K415031 cells fluoresce red. </center>
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Revision as of 07:33, 27 October 2010

Bacteria
the bacterial uv toggle

In the beginning, there was a UV Toggle (Kobayashi/Collins, 2004).

The 2010 MIT iGEM team saw that it was good, and decided to implement the Collins toggle in E.coli to create cells with bistable phenotypes. We planned for the toggle to create patterned fluorescence and phage polymerization in response to exposing the cells to UV light.


toggle by parts
toggle abstraction
toggle black-boxed
First Circuit

Collins Toggle Validation
We first wanted to make sure the parts we were working with were functional and reliable. As we wished to use the pTSMa plasmid as part of our circuit, we tested the original Collins toggle (pTSMa co-transformed with pCIRa).

Our recreation of the Collins toggle experiment validated pTSMa as a functional bistable toggle plasmid.
According to the paper, the area exposed to UV light should turn bright fluorescent green, and the rest of the cells should remain non-fluorescent. We recreated this experiment and our results demonstrate that the Collins toggle was working as it is described in the paper.

The basic Collins Toggle plasmids pTSMa and pCIRa. We used pTSMa in our own experiments as a bistable toggle.











Our First Biobrick
With our newly verified (borrowed) plasmid, we got to work on building our own response to UV induction. Using the same promoter used in pCIRa (the PcI-OR promoter that is induced by AHL/LuxR complex and inhibited by cI), we created a biobrick that would respond to UV induction by producing mCherry fluorescent protein when co-transformed with pTSMa. Our part would also constitutively produce LuxR, the protein that binds AHL and becomes a transcription factor that encourages the activity of the PcI-OR promoter.

Our first working part with a readout was K415006.

The first picture of mCherry fluorescence induced by UV light.





After much fine-tuning of the power of the UV exposure, the concentrations of AHL and IPTG, and mask cutting, a pattern of fluorescence finally emerged -- the first image.


Improved Bacterial Camera
As an intermediate in our project, we had created a bacterial camera with the ability to take and store images, reproduce the image on command (when AHL is added to the medium), and reset the entire plate to a blank slate whenever we pleased. All these abilities were a result of the bistable toggle we implemented in our design. In the context of past iGEM bacterial cameras, our toggle-based camera is a great improvement. UT-Austin created the first bacterial camera in 2005, exposing their cells to light for 12-15 hours to produce an image. Our toggle can snap and store an image in a matter of seconds.


AHL Propagation

We wanted our cells to be self-inducing and our signal to be self-propagating in addition to the circuit being sensitive and specific to user inputs. To do this, we added a part that would generate one of the necessary chemicals for transcription of the mCherry fluorescent protein.

Construction
We dubbed this part K415023, a part that now (if AHL and LuxR were available within the cell) fluoresced green or red in vivo depending on the state of the co-transformed toggle construct pTSMa. In addition, it would produce AHL under the same conditions as mCherry. This part can thus propagate and amplify a signal from a starting point of a droplet of AHL or even from leaky expression of AHL.

Movie
In order to view the propagation, we recorded a movie beginning from the moment the cells were induced with UV. In addition to viewing the progression of the fluorescence, we were able to record how long it took for a true pattern of fluorescence to emerge in our cell lawn.



Low Power Toggle

E.Coli population density as a function of UV exposure. Measured by drip assay.
According to established protocol, we were switching the state of our toggle (pTSMa from Collins 2003) with 80 J/m^2 UV. We noticed however, that significant cell death accompanied this exposure, likely due to fatal mutations.

LPT Construction
Searching through literature, we happened upon evidence of a super-sensitive cI (the lambda repressor from the pTSMa) that would be much more easily cleaved in UV-exposed cells. Luckily, this change from wildtype cI to the sensitive cI took a single SDM round, and we produced pLPTa, the Low Power Toggle.
Through a single point mutation by SDM, the MIT iGEM team was able to change pTSMa into pLPTa, a toggle that requires much less UV power to switch between states.












Improved Toggle
In order to truly understand the improvements we made in the Collins toggle, we exposed cells transformed with either pTSMa and K415023 (our fluorescent output), or pLPTa and K415023 to varying levels of UV. We hypothesized that we would get the toggle to turn "on" at a lower UV power with pLPTa.



Biobricking the Toggles

We first received pTSMa in a non-Biobrick standard plasmid. Then, when we created our Low Powered Toggle from pTSMa, we used Site-directed Mutagenesis, so that it still maintained the nonstandard backbone. We decided that two stable, well characterized toggle switches would be useful to iGEM teams in the future, and we designed primers to add Biobrick sites to the ends of both the plasmids. We then switched the toggles into pSB1C3 for entry into the Parts Registry and they now exist under the names BBa_K415300 and BBa_K415301.


Improving R0065

R0065 is a hybrid promoter that the MIT 2010 iGEM team used in our composite parts to control transcription of mCherry fluorescence. In conjunction with our toggle, pLPTa, this promoter would only allow expression of fluorescence in the presence of AHL, LuxR, and UV exposure. While we found the promoter very useful, it is described many times on its parts registry page as being "leaky". After looking into the promoter, we realized this was due to two -10 promoter sequences, possibly due to an error when designing the hybrid promoter.

In this FACS scatter, the x-axis represents green fluorescence, and the y-axis represents red fluorescence with zero UV exposure. Theoretically, there should be no red fluorescence. The red fluorescence in part K415023 is controlled by the original R0065, and in K415069 is controlled by our less leaky promoter K415031. You can see that the original promoter is more leaky than our fixed promoter: 3% of K415069 fluoresce red, while 15% of K415031 cells fluoresce red.