Difference between revisions of "Team:UESTC-China/Demonstrate"

In order to express multiple enzymes in E. coli, we firstly did the codon-optimized of the enzymes. And then these genes were commercially synthesized. Finally, with the application of Gibson Assembly and Golden Gate strategy, we successfully introduced the target gene into different E. coli expression vector. (Fig. 1)

No.	Vector	E. coli resistance	Description
1	piGEM2018-Module001	Amp	BBa_J23100-RBS-pelB+5D-Xyn10D-fae1A-RBS-pelB+5D-Xyl3A-RBS-pelB+5D-cex-RBS-pelB+5D-cenA-Ter
2	piGEM2018-Module002	Kan	Ter-Ter-RBS-Fdh-RBS-FRE_adhE-FRE_ackA-RBS-AtoB-RBS-AdhE2-RBS-Crt-RBS-Hbd-Ter
3	piGEM2018-Module003	Kan	BBa_J23100-RBS-FhlA-RBS-HydA-

Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful. (Fig. 2)

In order to verify whether E. coli BL21(DE3) with piGEM2018-Module001 has taken effect. We detect enzyme activity separately. (Fig. 3) Comparing with wild type, our E. coli carrying piGEM2018-Moudlu001 take effect. BL21(DE3) with piGEM2018-Module001 has the activity of ferulic acid esterase, xylanase and cellulase.

Meanwhile, we also use GC-MS to detect ferulic acid. The ferulic acid production was monitored by periodically taking samples from the supernatant of E. coli carrying piGEM2018-Module001. The ferulic acid production was monitored using the method of gas chromatography by periodically taking samples from the supernatant of fermented liquid, and the internal standard is carvacrol [3]. Ferulic acid will decompose into 9 carbon compounds in the case of ferulic acid in gas chromatography with nitrogen as carrier gas [4]. Fig.4 shows that the peak of ferulic acid derivative increased within 20 hours while ferulic acid derivative was not found in sample of wild-type E. coli. Fig.5 demonstrate that our product is ferulic acid by GC-MS.

To further find whether the cellulase had been expressed successfully in BL21 (DE3), the method of Congo Red assay was performed. The results are shown in Fig.6. The strains carrying piGEM2018-Module001 showed a zone of clearance created by hydrolysis of CMC showed that cellulase was successfully transcribed and translated by BL21(DE3). The empty vector control didn't show any zone of clearance around the colonies.

Work validation of multi-enzyme system in BL21(DE3) carrying piGEM2018-Module002. AtoB, Hbd, crt, ter and adhE2 catalyzed six-step reactions converting glucose to butanol. To test whether this multi-enzyme conversion system could work in our E. coli successfully or not, we detected the time production curve of butanol of BL21(DE3) carrying piGEM2018-Module002 under the anaerobic condition.

Besides, after a serious of butanol-production detection under different cultivating condition, we finally provided the necessary data for modeling. Then detecting the butanol production in 48h under the optimized conditions. (Fig. 8)And the final maximum yield of butanol is 0.37 g / L in 24 hours.

Furthermore, comparing with the production of butanol from other paper, our butanol production has reached a medium level. (Tab 1)

Source	butanol titer (g/L · h)	knocking other pathway
[7]	0.012 g/L · h	No
Our Design	0.018 g/L · h	No
[8]
[9]

To verify whether the piGEM-Module003 plasmid was working normally in E. coli DH5α. We set up an anaerobic fermentation unit. Because E. coli produces only carbon dioxide and hydrogen under anaerobic conditions, the removal of carbon dioxide theoretically allows us to produce relatively pure hydrogen. Then we test the collecting gas.

Then we test the collecting gas (Video 1). We can see from this video. The gas produced in turn a makes a sharp explosion when it meets the burning stick, while the gas produced in the wild makes the stick extinguish directly.

For quantitative assay, we measure hydrogen using gas chromatography under different cultivating condition to provide data for Modeling. Then detecting the butanol production in 48h under the optimized conditions. (Fig. 8)And the final maximum yield of hydrogen is xxx g / L in 48 hours.

The theoretical maximum yield of facultative anaerobic bacteria is 2 moles H2 per mole of glucose. Based on the optimum reaction conditions for the modeling guidelines, we tested the hydrogen content and rate of production in the 40-hour anaerobic fermentation gas. The end result is x moles of hydrogen per gram of glucose.

In the future, we will try to integrate the gene, which control butanol and hydrogen production. Therefore, our super E. coli could produce both butanol and hydrogen. [Fig. 11]

In the future, we will try to integrate the gene which control butanol and hydrogen production. Therefore, our super E. coli could produce both butanol and hydrogen.

During the production process, the accumulation of butanol will exert a great inhibitory effect on cell growth. Therefore, we will introduce the GroEL gene and GroES gene from clostridium acetone butanol to make E. coli heterologous expression of heat shock protein -- a molecular chaperone of GroESL, which has been shown to increase butanol production. Fig. 18 shows that GroESL have a good effect on butanol tolerance.

Due to limitations of experimental conditions, we are not able to knock out competitive pathway. However, many by-products will produce in the process of butanol production, namely frdABCD for succinate, ldhA for lactate, pta-ack for acetate, and adhE for ethanol (Fig. ). If we could knock out these competitive pathway, then our butanol production will be greatly improved.