Team:IIT Kharagpur/Parts
Overview
The gene involved in lycopene expression: crtEBI and autoinducer detector: luxR gene are cloned into pSB1C3. The final construct involves:
1. Part A: luxpR+reporter gene
2. Part B: luxpL+luxR
3. Vector backbone
Individual composite parts and the final construct are built using standard assembly method.
Standard Bio Brick Assembly Used
BBa_K274100 aka The Reporter Gene:
We are using the biobrick part - BBa_K274100 as our reporter gene. This part contains three other genes: BBa_K118014, BBa_K118006 and BBa_K118005. These three sub-parts are responsible for the enzymes CrtE, CrtB and CrtI respectively. This whole assembly is obtained on an ampicillin resistant plasmid pSB1A2. These three enzymes together form a red lycopene pigment with farnesyl pyrophosphate as a substrate. All these three genes have their respective ribosome binding sites before their sequence. This composite biobrick is put under constitutive promoter R0011 and arabinose-inducible promoter I0500 transformed and has been tested in E.coli strain MG1655.
BBa_K546003 aka The LuxR Gene
For the LuxR gene we have used the biobrick part - BBa_K546003. This whole part consists of LuxR + lux pR promoter which is regulated by the promoter lux pL. It acts as a binding site for RNA polymerase for the transcription of gene LuxR.k
BBa_R0062 aka The LuxpR Gene
The primary DNA sequence of interest contained in this bio-brick is a promoter activated by LuxR in concert with HSL. The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
BBa_S05321 Our Gene:
We are using the biobrick part - BBa_K274100 as our reporter gene. This part contains three other genes: BBa_K118014, BBa_K118006 and BBa_K118005. These three sub-parts are responsible for the enzymes CrtE, CrtB and CrtI respectively. This whole assembly is obtained on an ampicillin resistant plasmid pSB1A2. These three enzymes together form a red lycopene pigment with farnesyl pyrophosphate as a substrate. All these three genes have their respective ribosome binding sites before their sequence. This composite biobrick is put under constitutive promoter R0011 and arabinose-inducible promoter I0500 transformed and has been tested in E.coli strain MG1655.
Standard Assembly method
One of the biobricks is isolated by cleavage with restriction enzymes EcoRI and SpeI. The vector with second biobrick is linearized using double digestion at two very close sites of EcoRI and XbaI. After gel purification, the insert is then ligated into the linearized vector at sticky ends of EcoRI and compatible XbaI and SpeI.
Enzymes used for digestions are EcoRI, XbaI, SpeI and PstI.
