Revision as of 22:18, 27 September 2013

lux pL controlled luxR with lux pR controlled ndh (LVA-tag) coding for NADH dehydrogenase II

Description

This part is made up of a quorum sensing promoter (BBa_F2621) and ndh gene with a LAV-tag. Under control of the quorum sensing promoter, ndh gene will express periodically as the promoter is periodically activated and repressed. Since the ndh gene is described detailed in Part: BBa_K1036001, here is a detailed description of quorum sensing promoter. Firstly, we should be familiar with the quorum sensing mechanism, the luxI gene is at low expression level and produces LuxI protein that synthesizes a kind of acyl-homoserine lactone (AHL), which is a small molecule that can diffuse across the cell membrane and mediate intercellular coupling when it reaches the threshold as enough biomass accumulated. AHL will bind intracellular protein LuxR, which is also consecutively produced by luxR gene. The LuxR-AHL complex can activate the luxI promoter, and the positive feedback loop is built. However, since in this circuit the luxI gene behind the luxI promoter is changed into ndh gene (with LVA-tag), so it could only be activated by AHL produced by luxI gene on another plasmid, which contains Part: BBa_K1036003.

This year our team designed a synchronous oscillator which contained three main parts. The first part was pSB1C3-gfp-luxI which part was BBa_K1036003 with pSB1C3 backbone. The second one was pSB3T5-aiiA which part was BBa_K546001 with pSB3T5 backbone. The last one was pSB4K5-ndh which part was BBa_K1036000 with pSB4T5 backbone.

Experimental Data

Construction
The ndh gene was amplifed from the genome DNA of E. coli strain BL21(DE3) via PCR. Then RBS(BBa_B0034), LVA-tag and terminator(BBa_B0015) were fused with ndh via fusion PCR becoming RBS-ndh-LVA-TT marked as ndh. The DH5α colony carried ndh gene was sequenced after TA-cloning. The biobrick BBa_F2621(located in 2012-Plate 2-21F) digested by EcoR & XbaI and the ndh digested by EcoR & SpeI were ligated together by T4 ligase.The Fig.1 showed the result of pSB1A2-ndh construction and the changed backbone pSB4K5.


Expression
To test the expression of ndh gene, an experiment was designed. The strain E. coli BL21(wide type) carried plasmid pSB4K5-ndh was cultured in LB medium with 25 μg/ml kanamycin under the condition of 37^oC,200 rpm for 12 hours. Then the cultured medium was added into 100 volumes of flesh LB medium with 25 μg/ml kanamycin and cultured under the same condition. 10^-3 mM AHL was added into the medium after 2 hours and continued culturing for more 10 hours. The control was without AHL. The cell was harvested, washed twice by ddH₂O and ran in SDS-PAGE.

The result was showed in Fig.1 and ndh means without AHL and ndh+AHL means with AHL. The red and blue arrows showed the two up-regulation proteins, while the green arrow showed the down-regulation protein. The band showed by the orange arrow was assumed the ndh gene according to the protein size.

Sequence and Features