Part:BBa_K4390048
SELIS pbrR 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 | K4390039 |
| 2 | K4390042 |
| 3 | K4390044 |
| 4 | K4390005 |
Usage and Biology
This circuit comprises four parts which in order of left to right are pbrR regulated lambda cI expression, pbrR regulated mCherry expression, Lambda cI controlled CmR expression and pbrR expression construct.
pbrR expression construct (Part:BBa_K4390005) contains pbrR gene with constitutive promoter which is able to express pbrR in the cell continuously. PbrR is a repressor for lead resistance operons in bacteria. In gram-negative bacteria, pbrR can bind to the promoter of lead resistance operons which repress the expression. This repression activity is controlled by lead apperance. When lead is appeared in the environment, the pbrR will bind to lead ions which will no longer be able to bind to the promoter and release the lead resistance operons to be expressed (Borremans, B. et al., 2001).
pbrR regulated lambda cI expression (Part:BBa_K4390039) comprises a Lambda cI gene which is under regulation of pbrR 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, pbrR regulates Lambda cI expression by binding to the pbrR binding promoter which blocks the translation. Due to this, the expression of Lambda cI will only be activated when there are lead present.
pbrR regulated mCherry expression (Part:BBa_K4390042) comprises a mCherry gene which is also regulated by pbrR 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 lead is present, the binding of pbrR to lead releases it from binding with pbrR 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 pbrR biosensor. When there is no lead in the environment, the Lambda cI expression and mCherry expression will be repressed by pbrR binding with pbrR 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 lead in water. The present of lead will bind to pbrR which will stop pbrR 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 lead.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2579
Illegal NheI site found at 2602 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 84
Illegal SapI.rc site found at 975
Illegal SapI.rc site found at 1022
References
Borremans, B. et al. (2001) Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. Journal of bacteriology. 183 (19), 5651–5658.
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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