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	Vector map	Description
1	piGEM2018-Module004	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-Ter

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)

Xylanase can decompose xylan to xylose, and the activity of xylanase can be estimated by detecting the concentration of xylose.

Firstly, for quantitative assay, standard curves of ranging from 0-60mg/L Xylose were measured (Fig. 3). Then, we test the activity of xylan. Comparing with wild type, our E. coli carrying piGEM2018-Moudlu001 take effect. (Fig. 4)

Ferulic acid esterase can decompose ferulic acid p-nitrophenol ester to produce p-nitrophenol and ferulic acid. The changes of absorbance value under 410nm could be an indicator of changes in ferulic acid concentration. (Fig. 5)

To fully determine that our plasmid has taken effect. We 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. GC-MS was used to detect the concentration of ferulic acid using the method reported in another paper [1]. Ferulic acid will undergo this reaction and decompose into 9 carbon compounds in the case of ferulic acid in gas chromatography with nitrogen as carrier gas (Fig. 6). Fig.7 is chromatograms of the supernatant samples, the peak of ferulic acid derivative was observed from samples of tobacco carrying piGEM2018-Module001 after 0h and 24h. Most importantly, 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.8 demonstrate that our product is ferulic acid by GC-MS.

In order to find out if the cellulases had been expressed successfully in BL21 (DE3), the method of Congo Red assay was performed.

Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides CMC-Na but not short chain resulting in halo formation.

The results are shown in Fig.9. The strains carrying piGEM2018-Module001 showed a zone of clearance created by hydrolysis of CMC showed that cellulases was successfully transcribed and translated by BL21(DE3). The empty vector control didn't show any zone of clearance around the colonies.

To further detect the function of cellulases, we tested cellulases and cenA activity. Providing data for previously submitted parts ( BBa_K118022, BBa_K118022). Firstly, for quantitative assay, standard curves of ranging from 0-1000mg/L glucose were measured (Fig.10).

We measured the release of reducing sugar from filter paper by the 3,5-dinitrosalicylic acid (DNS) method for the cellulases activity.

The total amount of reducing sugars was expressed as glucose equivalents according to a standard curve prepared with glucose in the range from 0 to 1000 mg/L−1.

In addition, we measured the release of reducing sugar from CMC-Na with the 3,5-dinitrosalicylic acid (DNS) method for cenA activity. As shown in Fig 12, BL21(DE3) carrying piGEM2018-Module001 could Bacteria decompose CMC-Na while wild-type couldn't, which proved that cenA could work successfully.

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. Significantly, the peak of butanol increased within 24 hours while it was not found in sample of wild-type BL23(DE3).(Fig.13)

For quantitative assay, standard curves of butanol ranging from 1-5g/L were measured (Fig. 14). The butanol production was monitored using the method of gas chromatography by periodically taking samples from the supernatant of fermented liquid, and the internal standard is isobutanol. [2]

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

First, in order to verify that the gene expressed the protein in E. coli, we performed SDS-PAGE on the DH5α bacterial solution containing piGEM-Module003 and the control bacterial solution. FhlA has a brighter band at 97.4 kDa and Hyda has a brighter band at 42 kDa.

Next, it was verified whether the piGEM-Module003 plasmid was working normally in Escherichia 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.

Firstly, for quantitative assay, standard curves of ranging from 0-9ml hydrogen were measured (Fig. 17). Then, we measure hydrogen using gas chromatography. Fig.XX is chromatograms of the gas samples, the peak of hydrogen was observed from samples of DH5α carrying piGEM2018-Module003 after 0h and 24h. Most importantly, the peak of butanol increased within 24 hours while it was not found in sample of wild-type BL23(DE3).

We use gas chromatography (GC) to verify the gas produced by the fermentation unit. We injected three different gases, 5% hydrogen with 95% carbon dioxide, the gas produced by the DH5α fermentation of the piGEM-Module003 plasmid, and the gas after fermentation of the original DH5α strain. Fig

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.

Now, let’s listen to the beautiful sound.

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 Escherichia 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.

[6]Fiddler, W., Parker, W. E., Wasserman, A. E., & Doerr, R. C. (1967). Thermal decomposition of ferulic acid. Journal of Agricultural and Food Chemistry, 15(5), 757-761.

[7] Atsumi, S., Cann, A. F., Connor, M. R., Shen, C. R., Smith, K. M., Brynildsen, M. P., ... & Liao, J. C. (2008). Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic engineering, 10(6), 305-311.