Part:BBa_K4390045
SELIS arsR 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 | K4390038 |
| 2 | K4390041 |
| 3 | K4390044 |
| 4 | K4390002 |
Usage and Biology
This circuit comprises four parts which in order of left to right are arsR regulated lambda cI expression, arsR regulated mCherry expression, Lambda cI controlled CmR expression and arsR expression construct.
arsR expression construct (Part:BBa_K4390002) contains arsR gene with constitutive promoter which is able to express arsR in the cell continuously. ArsR is a repressor for arsenical resistance operons in bacteria. In gram-negative bacteria, arsR can bind to the promoter of arsenical resistance operons which repress the expression. This repression activity is controlled by arsenic apperance. When arsenic is appeared in the environment, the arsR will bind to arsenic ions which will no longer be able to bind to the promoter and release the arsenical resistance operons to be expressed (Wu, J. and Rosen, B. P., 1991; Diorio, C. et al., 1995).
arsR regulated lambda cI expression (Part:BBa_K4390038) comprises a Lambda cI gene which is under regulation of arsR 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, arsR regulates Lambda cI expression by binding to the arsR binding promoter which blocks the translation. Due to this, the expression of Lambda cI will only be activated when there are arsenic present.
arsR regulated mCherry expression (Part:BBa_K4390041) comprises a mCherry gene which is also regulated by arsR 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 arsenic is present, the binding of arsR to arsenic releases it from binding with arsR 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 arsR biosensor. When there is no arsenic in the environment, the Lambda cI expression and mCherry expression will be repressed by arsR binding with arsR 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 the arsenic in water. The present of arsenic will bind to arsR which will stop arsR 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 arsenic.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2535
Illegal NheI site found at 2558 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2720
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 978
References
Diorio, C. et al. (1995) An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria. Journal of Bacteriology. 177 (8), 2050–2056.
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.
Wu, J. & Rosen, B. P. (1991) The ArsR protein is a trans‐acting regulatory protein. Molecular microbiology. 5 (6), 1331–1336.
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