Difference between revisions of "Part:BBa K1919300"

Revision as of 02:44, 17 October 2016

livJ

LivJ encodes periplasmic binding protein for branched-chain amino acid ABC transporter which is encodes by operon livHMGF. The livJ gene product binds leucine, isoleucine, and valine. Please note that there is a short guide peptide on N terminal of livJ coding region, which is used to guide the precusor to be transported into periplasmic room. This device constructed in our team contains a promoter J23100, a RBS, livJ coding region and a terminator. The device can be transferred into E.coli and overexpress livJ directly. The expression of the livJ RNA in the part has been studied via qPCR.The quantified result suggests that compared with negtive control of E.coli (not cantain the plasmid), livJ has been highly expressed in E.coli BL21 and E.coli DH5a.

Sequence and Features

Quantitative PCR exams expression levels of liv operon

Liv operon (leucine isoleucine valine) contains six genes. We cloned all those genes into overexpression vector in which livJKHMGF are under control of promoter J23100. All those devices have been transferred into E.coli and RNA expression levels are scrutinized via qPCR. In the trial, we regarded two genes, rrsA and dnaA, as reference. The rrsA gene transcripts 16s RNA while dnaA encodes a component of DNA polymerase. The expression level of rrsA is very high whereas the dnaA expression level is quite low. Generally speaking, the expression level of gene we need to study is always higher than dnaA and lower than rrsA. The two reference genes expression level are relatively stable.

Figure 1: the physical position of six genes on genome of E.coli K12.

This figure shows all six genes’ physical position in liv operon. Those six genes participate directly in branched chain amino acid transport. Two periplasmic amino acid binding protein are encoded by livJ and livK. These two proteins confer specificity on LIV-I and LS transport system. The livJ gene product binds leucine, isoleucine and valine, whereas the livK gene product is specific to leucine. These two system share a set of membrane components, products of the livHMGF genes. LivH and livM are estimated two transmembrane proteins locateing in innermenbrane of E.coli. An analysis of the livH and livM DNA sequence suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The livG and livF proteins are less hydrophobic, but are also tightly associated with the membrane. Both livG and livF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins.

Figure 2: we presumed the folds of expression level of rrsA over dnaA are 10^10. Then the six genes expression level are calculated according to reference gene.

Figure 3: the data from figure 1 are collected to show again except for livJ. Since expression level of livJ is so high that other expression level of genes are difficult to look.

The figure 2 and 3 show different expression level of six genes compared to dnaA after transferring six overexpression vectors into DH5a and BL21 when presuming the folds of expression level of rrsA over dnaA are 10^10, that is, we normalized the folds of expression levels of rrsA over dnaA in not only BL21 but also DH5a to 10^10. Usually, normalization helps us to compare different levels under same background. Please note that the number in vertical axis do not reflects reality.

Figure 4: different expression level folds of rrsA over dnaA expression level in DH5a and BL21. T-test P<0.01

We found that expression level folds of rrsA over dnaA are very distinct (T-test, P<0.01) in BL21 and DH5a. The expression level of rrsA in DH5a is around 160,000 times higher than that of dnaA DH5a, while it is around 100,000 times in DH5a. Therefore, we take the distinct expression folds of rrsA and dnaA into consideration.

Figure 5: recalculate expression level after taking distinct expression level folds of rrsA over dnaA into consideration.

Figure 6: the data from figure 5 is showed again except for livJ since the livJ expression level is so high that overshadows others. In this figure, we can clearly recognize other genes’ expression level.