Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Xiaoyu Yu
Date created: 2014-10-15 11:00:00
Date modified: 2015-05-08 01:10:17
A Kill Switch with "memory" time repressed by IPTG
| Types | DnaRegion |
| Roles | engineered_region Composite |
| Sequences | BBa_K1405008_sequence (Version 1) |
Description
We plan to establish a new E.Coli strain without lacI and MazEF system gene to avoid the inference of cell itself. We will test cI background expression level to see if we need to knock out cI.
As is shown in Fig.1, Fig.2, Fig.3 and Fig.4, in the ???Stable State???, before poured into the soil, the E. Coli fertilizer is cultured in the medium with IPTG. Promoter 1 is a weak constitutive promoter, so lacI is transcribed and translated at a considerate low level. Therefore, high concentration of IPTG, which binds LacI and changes LacI???s conformation, is able to inactive LacI and open the promoter 2. Then, CI can be highly expressed to repress promoter3. Finally, mazF is inhibited.
After fertilizing, no more IPTG exists in the soil to bind LacI. As a result, LacI with a tag which stabilize the protein takes time to accumulate to a certain high concentration, which is the repression threshold of promoter 2, aiming to represses promoter 2 as well as the expression of cI. The time needed to finish this progress is the designed ???memory time???. During this process, E. coli takes its own responsibility to deliver Mo. And after that, it can be killed to reduce the pollution to environment.
As is shown in table 1, all the biobricks in the design are from the top 10 most used parts of iGEM to guarantee the feasibility.
Name Number Description
CI coding part BBa-C0051 Coding cI
Promoter 1 BBa-J23116 Weak constitutive promoter
Promoter 2 BBa-R0010 LacI repressible promoter
Promoter 3 BBa-R0051 CI repressible promoter
Table 1 Biobricks used in the design
In the future, we plan to model the ???memory time??? and do more wet-experiments to confirm its feasibility. As for the modeling, we will test the expression level of promoter 1, the minimal stand of LacI to repress promoter 2 and the degradation speed of LacI. On the other hand, we will test the background expression of CI after removing IPTG to check if the CI concentration is low enough to open the promoter 3. Then we will strive to make this system more effective.
Notes
1Source
Name Number DescriptionCI coding part BBa-C0051 Coding cI
Promoter 1 BBa-J23116 Weak constitutive promoter
Promoter 2 BBa-R0010 LacI repressible promoter
Promoter 3 BBa-R0051 CI repressible promoter
Table 1 Biobricks used in the design
| Access | Instance | Definition |
| public public public public public public public public public | BBa_J23116 BBa_K1088018 BBa_B0015 BBa_R0010 BBa_C0051 BBa_B0015 BBa_R0051 BBa_K302033 BBa_B0015 | BBa_J23116 lacI BBa_B0015 LacI cI lam BBa_B0015 cI lam BBa_K302033 BBa_B0015 |