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Project Description

Content

Team UC Santa Barbara's project goal is engineering a strain of E. coli to express GFP in response to contact with a different strain. The proposed mechanism of action is an application of contact-dependent growth inhibition (CDI). CDI is a system used by some bacteria to inhibit other bacterial cells that are in direct contact. Particularly, cells that do not have the proper immunity protein will be inhibited.

In applying CDI to fit the project goal, we are engineering two strains of E. coli: an "attacker" strain and an "indicator" strain. The attacker strain is engineered to express the CdiA-CT^UPEC536 C-terminal toxin fused to CdiA^EC93. The purpose of this is to emulate the presence of uropathogenic E. coli 536 (UPEC536) strain cells without requiring BSL-2 handling procedures (under CDI, this strain ideally functions identically to UPEC536). In order for the indicator strain to express fluorescent protein and withstand CdiA-CT^UPEC536, it expresses the following constructs: CysK-T25 (a permissive factor required for binding CdiA-CT^UPEC536 fused to a fragment of adenylate cyclase), T18-CdiI^UPEC536 (a complementary fragment of adenylate cycle fused to the immunity protein corresponding to CdiA-CT^UPEC536), and GFP under the pap promoter, which is cAMP-dependent, similar to the lac promoter. Once CdiA-CT^UPEC536 is inserted into an indicator cell, it binds to CysK and CdiI, bringing T25 and T18 in close proximity to function as an adenylate cyclase unit (principally based on the bacterial adenylate cyclase two-hybrid system (BACTH)). cAMP is thereby produced, inducing the expression of GFP via the pap promoter. Notably, the indicator strain is cyaAΔ so that cAMP production is only caused by the joining of T25 and T18.

References

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