Team:CCU Taiwan/Composite Part

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comDE two components signal transduction system

This is a composite part used for producing comD, comE, two essential protein for the CSP two components signal transduction system. Originated from Streptococcus mutans. ComD is a kinase and membrane-associated receptor. After comD binds to the quorum sensing molecule CSP, the comD-CSP complex will phosphorylate comE. Then, the phosphorylated comE can turn on the transcription of downstream gene. FLAG-TAG is added after comE and c-Myc is added after comD.


CSP detection through GFP intensity

This is the part that we used to detect one of our target molecule, CSP. It will produce membrane receptor, comD. ComD then will bind to CSP. The comD-CSP complex will phosphorylate comE. The phophorylated comE, finally will bind to a comE responsive promoter (on this part) and produce GFP. Through GFP intensity, we will learn the concentration of CSP.


Lactate detection

This construction is used for lactate quantization We made use of the property of LldR operon. The LldR is transcription factor. Without lactate, it will bind on O1&O2 operator site, form a DNA loop, blocking the transcription. As lactate present, lactate will bind on LldR and open the loop, allowing the transcription to go on and produce GFP. GFP intensity is then detected and deduct lactate level. This construction is cloned into E. coli.


MazEF kill switch

MazEF is a suicide system in Escherichia coli which consists of a stable toxin mazF and labile antitoxin mazE. MazF causes cell death by cleaving mRNA at a specific site. On the other hand, mazE plays a role of antitoxin which can antagonize the toxin mazF.
In general, continuous generation of MazE can prevent the situation of cell death by antagonizing mazF. However, once mazE ceased to be produced, it would be degraded by the protease ClpAP. Hence, the remaining toxin mazF will cause the cell death by cleaving mRNA.
This is a composite of mazEF that is regulated by arabinose. When arabinose is added, mazEF will be produced. And once arabinose supply is in short, mazE ceased to be produced, it would be degraded by the protease ClpAP.