Device

Part:BBa_K1919300

Designed by: Shaobo Yang   Group: iGEM16_SCU-China   (2016-09-16)
Revision as of 06:56, 17 October 2016 by Shaobo (Talk | contribs)


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 382
    Illegal AgeI site found at 203
    Illegal AgeI site found at 485
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 366


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.

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data process 1: presume folds of expression level of rrsA over dnaA are same in BL21 and in DH5a

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.



data from reference genes suggested folds of expression level of rrsA over dnaA are distinct in BL21 and DH5a

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.



data process 2: take discrepancy of folds of expression level of rrsA over 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.

Please note that negative control set are not shown in the figure, since all of expression levels of six genes in wild BL21 or DH5a are below 1 in the figure. Albeit the DH5a owns livJKHMGF in genome, the expressions of them are repressed by a regulator, Lrp (leucine-responsive regulator protein). The number in vertical axis indicates how many times the expression level of certain gene is higher than that of dnaA. We assume that the expression level of danA is 1 in BL21 and DH5a, then the expression level of livJ, for example, is 2339 in DH5a and 9304 in BL21. As we can see from figure 4 and figure 5, livJ and livK are the two highest expressed genes, especially for livJ. And there is a tendency that expression level of genes reduces in accord with physical position on genome from left to right. We believe it reflects the function of those genes to some extent, since there need more periplasmic binding protein (livJK) than receptors (livHMGF) on membrane so that uptake of amino acid will be more efficient.



Leu absorption trial

novel protocol for leucine concentration examination

To exam which overexpression vector can promote leu uptake most drastically, we transferred all six genes into BL21 and DH5a. We designed a novel protocol to exam fluctuation of leu concentration in liquid LB medium. The protocol goes that:

Materials that should be prepared in advance

  • 1 sulfosalicylic acid solution 20 g solid sulfosalicylic acid in dissolved in 100ml ddwater
  • 2 ninhydrin solution 1 g ninhydrin is dissolved in 35ml ddwater
  • 3 pH8.03 PBS 
A solution  4.5350g KH2PO4 is dissolved in 500ml ddwater
B solution  11.938g Na2HPO4 is dissolved in 500ml ddwater
 pH8.03 PBS = 10ml A solution +190ml B solution

procedure

  • 1 1ml sterilized LB medium is inoculated with microbe in 1.5 ml EP tube and shaking in 37 ℃ overnight
  • 2 10000g*1min for centrifugation and then pipette 800ul supernatant to another new 1.5 ml EP tube
  • 3 pipette 300 ml LB to other two new 1.5 ml tubes and add 100ul sulfosalicylic acid solution
  • 4 shaking violently for 1 min
  • 5 20000g*10min and then pipette 300ul supernatant to a new 1.5 EP tube
  • 6 add 1ml pH8.4 PBS buffer and mix them by reversing the tube several times
  • 7 all 0.15ml ninhydrin solution and mix them by reversing the tube several times
  • 8 incubate the tube in room temperature for 1hr
  • 9 detect the optical density via spectrophotometer


full wavelength scanning

Figure 7: full wavelength scanning of liquid LB implemented with leucine (A) or without leucine (B). We detected that there is a high peak of absorption in 570 nm.

Before further test, we exam two kinds of liquid LB medium that are with or without additional leucine implement. The liquid medium was processed follow the protocol above and tested by spectrophotometer for full wavelength scanning from 300nm to 1000nm. Since liquid LB medium teems with amino acid, short peptide and other nutrition, we can see a peak in B (without leucine implementation) 570nm as well.


optical density at 570nm

Figure 8: liquid mediums shaking with DH5a harboring different overexpress vectors are tested by spectrophotometer at 570nm.

The optical density as 570nm of liquid LB implemented with leucine. Nctr is a negative control set that do not contain microbe. 2101 is a negative control as well but inoculates with an E.coli named 2101 that harbors chloromycetin resistance but only expresses a short non-coding RNA. LivJKHMGF indicates medium after shaking overnight with E.coli DH5a contain corresponding overexpression vectors. The data of BL21 is not shown here since the data of different sets only owns tiny discrepency. We try to explain these results, but we are not sure the following explanation reflects reality to what extent. To ascertain this question, there need more sophisticated investigation. As we have mentioned before, liv JKHMGF are not resides on genome of Bl21. In addition, the leucine ABC transporter will not work if there is not other component expressed in cell. Thus, it can be understood that there are tiny discrepency among different sets of experiments in BL21. For DH5a, Albeit the DH5a owns livJKHMGF in genome, the expressions of them are repressed by a regulator, Lrp (leucine-responsive regulator protein). According to the result of BLAST, Lrp not only resides on genome of DH5a but that of BL21. Lrp is a dual transcriptional regulator for at least 10% of the genes in Escherichia coli. These genes are involved in amino acid biosynthesis and catabolism, nutrient transport, pili synthesis, and other cellular functions. It is believed that Lrp senses the presence of rich nutrition based on the concentration of leucine and positively regulates genes that function during famine and negatively regulates genes that function during a feast. Lrp-leucine DNA bingding transcriptional dual regulator inhibits livJ transcription initiation, whereas L-leucine binds to Lrp transcriptional dual regulator to block inhibition of livKHGMF. As shows in figure 8, livJK seem do not increase uptake of leucine compared with 2101, livHM inhibit uptake of leucine compared with 2101 and livGF promote uptake of leucine dramatically, especially for livF. We can brace these six genes into 3 group, livJK, livHM and livGF, according to the result of spectrophotometer. Then we attempt to ascertain the inner relationship that brace these genes into the 3 group.



explanation of optical density data

affection of leucine in coding region

Figure 9: number of leucine in these six genes.
Figure 10:percentage of leucine in these genes =number of leucine/number of amino acid

Firstly, we list leucine number and percentage in these genes, and we considered that if there are more leucine in the protein, it will uptake more leucine from medium. But we did not find any relevance between these data and results in figure 8.

Then we scrutinize the construction of the leucine ABC transporter. Although there is a little information for the transporter, we find that the hydrophilicity plots can brace the six genes into three categories.

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