Part:BBa_K4390047
SELIS mutated merR evolution construct
This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.
This is a level 2 part formed by assembly of the following level 1 parts:
| 1 | K4390040 |
| 2 | K4390043 |
| 3 | K4390044 |
| 4 | K4390003 |
Usage and Biology
This circuit comprises four parts which in order of left to right are mutant merR regulated lambda cI expression, mutant merR regulated mCherry expression, Lambda cI controlled CmR expression and mutant merR expression construct.
mutant merR expression construct (Part:BBa_K4390003) contains mutant merR gene with constitutive promoter which is able to express mutant merR in the cell continuously. Mutant merR contains several mutations which convert it to be more specific to cadmium. Mutant merR works similar with merR with repression activity when binding to mutant merR regulating promoter. This repression activity is controlled by cadmium apperance. When cadmium is appeared in the environment, the mutant merR protein will undergo conformational change which will no longer be able to bind to the promoter and release the cadmium resistance operons to be expressed.
mutant merR regulated lambda cI expression (Part:BBa_K4390040) comprises a Lambda cI gene which is under regulation of mutant merR regulating promoter. Lambda cI is a transcriptional repressor which allows Lambda phage to establish and maintain latency after infect E. coli. It regulates the entry of lytic cycle by repressing the lytic promoters (Johnson, A. D. et al., 1979). This Lambda cI sequence was codon optimised for expression in E. coli K12, and was be used in Seamless Enrichment of Ligand-Inducible Sensors (SELIS) as the repressor (d’Oelsnitz, S. et al., 2022). In this part, mutant merR regulates Lambda cI expression by binding to the mutant merR binding promoter which blocks the translation. Due to this, the expression of Lambda cI will only be activated when there are cadmium present.
mutant merR regulated mCherry expression (Part:BBa_K4390043) comprises a mCherry gene which is also regulated by mutant merR regulating promoter. mCherry is a red fluorescent protein which derived from DsRed of Discosoma (Shaner, N. C. et al., 2004). This protein will generate bright red colour when expressed which is always been used as reporter in research. In this part, when cadmium is present, the binding of mutant merR to cadmium releases it from binding with mutant merR binding promoter, which will activate the expression of mCherry and the colony will appear red.
Lambda cI controlled CmR expression (Part:BBa_K4390044) contains a CmR gene and a Lambda cI regulated promoter. CmR gene encodes protein which is able to induce chloramphenicol resistance by triggering putative efflux pump (Nilsen, I. W. et al., 1996). In this part, the expression of CmR will only be activated without Lambda cI, which is used for selecting the colonies that contains functional construct.
This whole circuit is designed to improve the accuracy and specificity of mutant merR biosensor. When there is no cadmium in the environment, the Lambda cI expression and mCherry expression will be repressed by mutant merR binding with mutant merR regulating promoter, result wild-type colour and no Lambda cI presenting. This will release the activation of Lambda cI controlled CmR expression and generate chloramphenicol resistance. This property can be used to select the colonies that contains the functional construct after assembly and transformation since the bacteria that does not contain functionally construct will die on chloramphenicol plates. After the functional constructs are obtained, it can be used to test cadmium in water. The present of cadmium will bind to mutant merR which will stop mutant merR from repressing Lambda cI expression and mCherry expression. The Lambda cI expression will regulate the expression of CmR gene, therefore the cells will lost the chloramphenicol resistance. The expression of mCherry will bring red colour which can be observed easily, which can is be used as a reporter for cadmium.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2505
Illegal NheI site found at 2528 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2811
Illegal AgeI site found at 2913 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 948
References
D'Oelsnitz, S. et al., (2022) Using fungible biosensors to evolve improved alkaloid biosyntheses. Nature chemical biology. 18 (9), 981–989.
Johnson, A. D. et al. (1979) Interactions between DNA-Bound Repressors Govern Regulation by the $\lambda $ Phage Repressor. Proceedings of the National Academy of Sciences - PNAS. 76 (10), 5061–5065.
Nilsen, I. W. et al. (1996) Isolation of cmr, a novel Escherichia coli chloramphenicol resistance gene encoding a putative efflux pump. Journal of Bacteriology. 178 (11), 3188–3193.
Shaner, N. C. et al. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature biotechnology. 22 (12), 1567–1572.
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