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Team:Paris Bettencourt/Delay
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===Objectives=== | ===Objectives=== | ||
| - | As an alternative delayed trigger for our suicide machinery we sought to make use of the natural delay experienced by cells as they approach stationary phase. | + | As an alternative delayed trigger for our suicide machinery we sought to make use of the natural delay experienced by cells as they approach stationary phase. The design requirements for this system are similar to those described above. |
| - | * | + | *The delay must be programmable and sufficiently long. |
| - | + | *Death must follow inevitably one the countdown begins. | |
| - | * | + | *The suicide machinery is highly toxic, and must not be expressed until needed. |
| - | + | ||
| - | * | + | |
===Design=== | ===Design=== | ||
Revision as of 00:17, 27 September 2012
- Main
- Team
- Project
- Achievements
- Human Practice
- Safety
- Notebook
- Attributions
Overview
Delay system
Semantic containment
Restriction enzyme system
MAGE
Suicide system
Encapsulation
Synthetic import domain
Safety Questions
Safety Assessment|
Aim : A programmed delay will allow the cell to perform its intended function before our DNA-degrading suicide machinery is expressed. Experimental system: We used two different approaches to create this delay. The first one is based on the gradual dilution of a regulatory transcription factor. The second one makes use of a stationary-phase specific promoter. Both systems eventually result in the expression of the restriction enzyme I-SceI. In the final design, I-SceI cleaves the antitoxin gene, ultimately dooming the cell. Each step in this causal sequence contributes to the overall delay in the system.
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Contents |
Overview
The delay system serves to suppress the function of the suicide device and preserve the integrity of the host genome while the organism does its intended job in the environment. We experimented with two designs for producing a programmed delay before the expression of the DNA-degrading suicide machinery.
The dilution delay system relies on the lab-specific expression of a transcriptional inhibitor. This inhibitor is induced by a specific compound found in the laboratory but not in the environment. When the cell enters the environment the inhibitor is gradually degraded or diluted by cell growth. Eventually, the repressor concentration falls below a critical threshold, releasing the suicide machinery.
The sRNA delay system makes use of a stationary-phase specific promoter. Under laboratory conditions, the suicide machinery is repressed by an inducible sRNA. In the environment, the suicide machinery is expressed as soon as the cells reach stationary phase.
Dilution delay system
Objectives
Our cells need time to work in the environment before we degrade their genomes. A programmed delay circuit faces the following challenges:
- The delay must be programmable and long enough for our cells to perform a useful task.
- Once begun, the delay countdown should lead inevitably to death, and should not be reversible.
- Prior to induction, our suicide machinery must be very tightly repressed.
Design
We chose to base our delay on a simple transcriptional network. The arabinose-activated promoter, pBAD, drives the production of the LacI gene. LacI represses a restriction enzyme at the pLac promoter. In the absence or arabinose, the restriction enzyme is eventually expressed, triggering irreversible cell death.
We chose the pLac promoter because of its reputation for tight repression by the lacI protein. Our final design eventually expresses a restriction enzyme, because the DNA hydrolysis reaction is effectively irreversible. But for the purposes of characterization, we substituted GFP for the restriction enzyme at the end of our transcriptional cascade.
The system we created to characterize our delay is shown below:
Characterization
We characterized the behavior of our transcriptional circuit in the presence of IPTG and/or arabinose, or without external inducers (control). GFP expression is induced by IPTG and repressed by arabinose, as expected. By growing the cells in the presence of arabinose and then removing it, we expect to be able to quantify the delay in GFP expression created by our system.
sRNA delay system
Objectives
As an alternative delayed trigger for our suicide machinery we sought to make use of the natural delay experienced by cells as they approach stationary phase. The design requirements for this system are similar to those described above.
- The delay must be programmable and sufficiently long.
- Death must follow inevitably one the countdown begins.
- The suicide machinery is highly toxic, and must not be expressed until needed.
Design
We want to use a sRNA as a way to block the expression of the toxin. The concept of our design is described below. Similar systems have been shown to respond in a treshold-linear way [2]
In order to achieve our goal, we started form the construct of Yokobayashi et al. [1] described below.
Even though the cloning is still in process, we hope to build the following construct by the end of the competition.
The transcription of the toxin is controlled twice : first, we chose a stationary phase promoter, yiaGp. It is known be recognized by the sigma-S subunit of the RNA polymerase, and not the exponential phase sigma-70 subunit. [3], [4].
We have shown that the yiaGp promoter is indeed activated during the stationary phase in our construct, even though the level of trancription is rather low (see experiments)
In order to achieve a complete lockdown of the toxin, we added a second repression mechanism, this time at the post transcriptional level. We used and modified the constructs of Yokobayashi et al. to allow a repression of the translation using sRNA [1]. We chose to use the sRNA 7.9, as it has been shown to bind the leader sequence of its target mRNA and not its coding sequence, and to allow a 20 fold repression of the protein’s expression. The transcription of the sRNA is under the control of the pBAD promoter. The coding sequence of colicin E2 is fused to the leader sequence (5’ UTR) of OmpF. The araC protein allows us to use this construct in cells deleted for the arabinose operon (TOP10). We hope that, using this type of cells, the delay will depend on the dilution of the arabinose in the medium as it is not metabolized. We will need to check the effect of extracellular glucose on the level of repression, as the CRP protein is known to repress the pBAD promoter.
Characterisation of the YiaGp promoter
Using the following construct, we characterized the stationary phase YiaGp promoter in TOP10 cells.
YiaGp is a late stationary phase promoter. It has been shown to be activated by 10 to 30 fold during to stationary phase, and is among the strongest promoters during this phase. Furthermore, its action has been reported to last for a longer time than most stationary phase promoters.
Experimental setup
Results
Present your results
Refined characterization of the Yokobayashi et al. sRNA repression plasmidic device
We double transformed TOP10 cells with the pKP33-GFPuv and pBAD-aOmpF-7.9 plasmids (see [1] for details, and click here for a schematic of the genetic system). We chose the 7.9 sRNA because it seemed to show a strong repression of the GFP expression, and bind to the RBS of the aOmpF leader sequence and not to the coding sequence.
The cells were grown in LB until stationary phase, then diluted 100-fold in LB. After adding various arabinose concentrations to the medium, we monitored the fluorescence of GFP (excitation : 395nm, emission : 509nm) using a TECAN 96-wells plate reader during 10 hours . We had 6 replicates for each concentration. The bar plot below shows the average normalized fluorescence at the final time point for each concentration tested.
References
1. Sharma, V., Yamamura, A., & Yokobayashi, Y. (2012). Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli, 6-13. [http://pubs.acs.org/doi/abs/10.1021/sb200001q| Paper]
2. Levine, E., Zhang, Z., Kuhlman, T., & Hwa, T. (2007). Quantitative characteristics of gene regulation by small RNA. PLoS biology, 5(9), e229. doi:10.1371/journal.pbio.0050229 [http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0050229| Paper]
3. Sharma, U. K., & Chatterji, D. (2010). Transcriptional switching in Escherichia coli during stress and starvation by modulation of sigma activity. FEMS microbiology reviews, 34(5), 646-57. doi:10.1111/j.1574-6976.2010.00223.x[http://jb.asm.org/content/186/21/7112.long| Paper]
4. Shimada, T., Makinoshima, H., Ogawa, Y., Miki, T., Maeda, M., & Ishihama, A. (2004). Classification and Strength Measurement of Stationary-Phase Promoters by Use of a Newly Developed Promoter Cloning Vector, 186(21), 7112-7122. doi:10.1128/JB.186.21.7112 [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2010.00223.x/abstract;jsessionid=8C98B1B383242B9DFF45429802F48428.d02t04| Paper]
