On this day, our team was formally established. BIT-China for one green earth is our slogan .And what we want to do is to improve the optimistic temperature from 37℃ to 42℃ in fermentation of E.coli ,then we can contribute to increase the competitive of fermentative products with many aspects, including reducing the consumption of energy of source, simplifying productive process and improving the efficiency.

We design two systems to reach our goal. The first system is the customize thermotolerance system. We choose heat shock protein family (HSP) with the control of RNA thermometer. Another system is intelligent quorum regulating system, we use quorum sensing device and programmed cell death(PCD) device to control the cell density ,aiming at maintaining cell in an optimistic cell density to control the release of heat.

We optimized our design, and choose the red fluorescent protein as the goal products.

According to our goal, we designed some new genes chosen some known genes. Following figure is a brief gene line.

Combining the references and experimental verification, we finish the design of whole pathway. The following figure shows the detail information.

HSP:
We amplified these genes respectively by PCR, including GroeS、GroeL、DnaK, DnaJ, ThiF, TTE, IbpA, FliA, RpoE3 and RpoE7. Then we connected these genes with Gapa promoter in plasmid PET-28a and transformed it into E.coli.

HSP:
This week, we finished the work of GroeS, GroeL, DnaK and DnaJ. Here are the results by Colony PCR.

This week, we completed the work of ThiF, TTE, IbpA, FliA, RpoE3 and RpoE7. The results of verification by Colony PCR are shown as follow.

HSP:
From this week, we began the fermentation of our engineering strains. This week we verified the function of these genes with the temperature of 40℃.

HSP:
Fermentation was conformed in 43℃ during this week.

We verified the function of these genes in 46℃ by fermentation.

After being compared with each other, we used GroeS, GroeL, DnaK, DnaJ and ThiF to make heat resistance part. We began on the work of verifying the function of these genes in Saccharomyces cerevisiae. We changed the promoter into FBA1 promoter which is a strong one. After transforming the plasmids with targeting genes into Saccharomyces cerevisiae successfully, we got our engineering strains. Currently, we finished the fermentation of TTE strain.

This week, we worked mainly on the construction of standard heat-resistance part. According to our design, we connected a RNA thermometers named ROSE and FourU with targeting genes. Then we combined these genes with plasmid PET-28a and transformed it into E.coli.

PCD:
We cloned mazeE and mazF from genome of BL21/DE3. The length of maze gene was 249bp. We could see a series of bands around 250bp. We found the optimal annealing temperature in PCR was 57 degree. On the other hand, the length mazF gene was 336bp. The bands were located between 250bp and 500bp. The optimal annealing temperature was 57 degree, too.

Quorum sensing

we connected B0034 with C0061

B0034 and C0062 are fitted together

B0034 and C0040 are fitted together

PCD:

We transformed BBa-1763007: promoter lambda, which is regulated by cI, with RFP reporter. The length was 918bp.

The total length is 1,223bp (918 + 305). On the gel, we saw a series bands around 1,000bp, some of which were very bright.

Quorum sensing:

We linked J23102/J23119/J23110/J23118 to B0034-C0062 respectively

We connected J23119 with B0034-C0062

We connected R0063 with B0034-C0040

PCD:

We ligated PcI with eGFP trough OE PCR.

First one was the ligation of PcI and RBS, the length is 10+75+39=124bp
The second one was eGFP, whose length is 39+720+29=788bp
The third one was the ligation of the three(PcI-RBS-eGFP) , the length is 10+75+720+29=834bp.
All the results could be observed on gel, showing our work didn’t have mistakes. All the optimal temperature in PCR is 57 degree.

Quorum sensing:

We linked B0015 to (J23102/J23119/J23110/J23118)-B0034-C0062

We linked B0015 to J23119-B0034-C0062

We connected B0015 with R0063-B0034-C0040

PCD:

We used EcoRI and SalI to digest the PET-28-a plasmid and pCI-eGFP and ligated both together.

Quorum sensing:

We joined (J23102/J23119/J23110/J23118)-B0034-C0062-B0015 to J23119-B0034-C0062-B0015

We joined R0063-B0034-C0040-B0015 to I13521

PCD:

We transformed the pCI-eGFP and verified the activity of pCI by FCM. Compared with CK group, EG group showed a higher activity. Then we compared the promoter activity. After the comparison, we chose the pyka and pgi as the mazF’s constitutive promoter.

Quorum sensing:

We linked (J23102/J23119/J23110/J23118)-B0034-C0062-B0015 to J23119-B0034-C0062-B0015 to R0063-B0034-C0040-B0015 to I13521, all the parts of quorum sensing device have been fitted together.

PCD:

We transformed Part:BBa_I719005(23 bp) T7 Promoter with Part:BBa_P0451(930 bp) CⅠ lam .I ligated T7 Promoter and CⅠ lam through 3A assembler. The ideal band, which is around 1,000bp can be seen on the gel.

Quorum sensing:

We verified the function of quroum sensing device

Oscillating circuit:

R0011 and B0034-C0040 are fitted together

We linked R0040 to P0451

B0034 and C0012 are fitted together

PCD:

We ligated pyka with mazF and ligated pgi with mazF through OE PCR. Then length of pykA with mazE is 127+336+32=495bp. Length of pgi with mazE is 524+336+32=892bp. On the gel, bands were showed around 500bp and 1,000 bp, proving we do that correctly.

Oscillating circuit:

We joined R0051 to B0034-C0012

We connected R0011-B0034-C0040 with B0034-C0062-B0015

PCD:

We used IPTG as our inducer, examining the OD of E.coli with MazEF system. We saw apparent growth inhibition from EG group, whose expression of MazE was inhibited. Then our PCD device could be proved that it really worked.

Oscillating circuit:

We linked R0011-B0034-C0040-B0015 to R0040-P0451

We linked R0051-B0034-C0012 to B0015

PCD:

We examined OD of normal E.coli and E.coli with exogenous HSP, DnaK. The result showed that HSP expressed in relatively cool condition did have apparent negative affection on the growth of E.coli. But as the time passed, the OD were relatively close. We thought that HSP worked and helped to folded incorrect proteins, then the growth of cell were contributed.

Oscillating circuit:

We joined R0011-B0034-C0040-B0015-R0040-P0451 and R0051-B0034-C0012-B0015 together. Then all the parts of oscillating circuit device have been fitted together.

We combined all of the devices together and verified its function by fermentation experiment.