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

Line 452: Line 452:
 
 
 
//滚动730px，左侧导航固定定位
 
//滚动730px，左侧导航固定定位
−
if ($(window).scrollTop()>300) {
+
if ($(window).scrollTop()>350) {
 
$('.fl_l').css({'opacity':'1'})
 
$('.fl_l').css({'opacity':'1'})
 
}else{
 
}else{

Revision as of 14:38, 14 October 2018

team

Demonstrate

  • Pathway construction
    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
    Figure 1. The introduction of piGEM2018-Module001 to piGEM2018-Module003.
    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)
    Figure 2.  The image of agarose gel electrophoresis by double enzyme digestion.
    (a)piGEM2018-Module001 (Line1,enzyme digested by EcoR32Ⅰ+NcoⅠ; Line2,enzyme digested by NcoIⅠ+XhoⅠ)
    (b) piGEM2018-Module002 (Line1,enzyme digested by PstⅠ+KpnⅠ; Line2,enzyme digested by HindⅢ+KpnⅠ)
    (c)piGEM2018-Module003 (Line1,enzyme digested by EcoRⅠ+NcoⅠ；Line2, enzyme digested by BamHⅠ+BglⅡ)
  • Three pathway validation
    Straw degradation and glucose production
    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)
    Figure 3.  The standard curve of Xylose.
    Figure 4. Two groups of different sample solutions were added. Namely, Module001:
    supernatant of piGEM-Module001. WT: supernatant of Wild Type. The strain BL21 (DE3) transformed with piGEM2018-Module001 was cultivated overnight and centrifuged to obtain the supernatant. The remaining bacteria were broken and broken products were obtained.1 ml xylan solution was added to 5 ml phosphate buffer solution (pH=6), incubated at 40 C for 5 min, and 1 ml sample solution was added.
    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)
    Figure 5.  The strain BL21 (DE3) transformed with piGEM2018-Module001 and wild type were broken. DSMO solution of ferulic acid p-nitrophenol ester was added to phosphate buffer solution of 630 L pH=6.4 with a concentration of 400 L of 10 mmol/L. Heat preservation 5min at 40℃ and add 0.2ml sample solution. The initial OD were same. Gently mix and incubate 4h at 40 C, determine the final OD value and compare the difference.
    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.
    Figure 6. 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.
    Figure 7. Chromatogram of Ferulic Acid derivatives and Internal Standard carvarol in supernatant of transgenic E. coli carrying piGEM2018-Module001 and wild-type E. coli. Samples were taken from reaction mixture at 0h, 24h.
    Figure 8. Mass spectrums of ferulic acid derivative. Sample was transgenic E. coli carrying piGEM2018-Module001 and taken from reaction mixture at 24h.
    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.
    Figure 9. Line 1: BL21(DE3) carrying piGEM2018-Module001 （OD600=1, 3, 5 from left to right) Line 2: Positive control enzyme (concentration=0.2, 0.3, 0.4 mg/ml from left to right) Line 3: BL21(DE3) carrying empty vector control (OD600=1, 3, 5 from left to right)
    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).
    Figure 10. The standard curve of Glucose.
    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.
    Figure 11. Reactions were carried out in 50 ml centrifuge tubes with 50 mg of filter paper (1 cm × 6 cm piece) in 1.0 ml potassium phosphate buffer (50mM, pH 7.0), plus 0.5 ml of the crude enzyme solution. Reaction mixtures were incubated at 40 °C for 3 h, enzyme action was interrupted by the addition of 3,5-dinitrosalisylic acid (DNS) reagent, which was used to quantify the total amount of reducing sugars. Reaction mixtures were then placed in a boiling water bath for 5 min, cooled to room temperature and diluted to 25 ml with water for the measurement of absorbance at 540 nm with a spectrophotometer.
    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.
    Figure 12. The concentration of reducing sugar after 3 hours. Reaction proceeded in 50 mM potassium phosphate buffer (pH = 7.0) at 40°C. Module001: The crude enzyme solution obtained by ultrasonication of BL21(DE3) carrying is involved in the reaction. Wild Type: The crude enzyme solution obtained by ultrasonication of wild-type BL21(DE3) participated in the reaction. CMC-Na:The same amount of CMC-Na participated in the reaction as a control. Each data represents the mean value ± standard deviation from two independent experiments.
    Butanol production
    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)
    Figure 13. Chromatogram of butanol and Internal iosbutanol of transgenic E. coli carrying piGEM2018-Module002 and wild-type E. coli. Samples were taken from reaction mixture at 0h, 24h.
    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]
    Figure 14. Standard curve of Butanol.
    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.
    Figure 15.  The butanol production curve at optimized conditions (temperature=34℃, initial OD=13, Initial pH=8)
    Hydrogen production
    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.
    Figure 16.  M: Marker, WT: wild type, Module 003: piGEM2018-003 in DH5α. All samples were obtained after breaking the cultivating E.coli DH5α for 4h at 37℃, 180rpm.
    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.
    Figure 17.  The left side of the apparatus is a carbon dioxide generating apparatus, and carbon dioxide is produced by dropping a dilute hydrochloric acid into a flask containing CaCO3 under the separatory funnel. The middle part of the device is a bacterial liquid fermentation device consisting of a three-necked flask placed on a temperature-controlled magnetic stirrer. The magnetic stirrer can continuously stir the bacteria liquid and keep the temperature constant. The right side of the unit consists of two scrubbers and a sink. The first scrubber bottle is filled with a sodium hydroxide solution to remove carbon dioxide and hydrogen chloride gas from the mixed gas. In the second scrubber is a clarified lime water used to verify that the carbon dioxide gas has been removed. The final product gas is then collected using a drainage gas collection method in the water tank.
    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).
    Figure 18.  Standard curve of Hydrogen.
    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
    Figure 19. 
    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.
    Figure 20. 
    Now, let’s listen to the beautiful sound.
    video
  • Work going on
    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.
    Figure 21.  After adding 0.3ml butanol in 30ml LB medium, the measurement of optical density (A600) deference between DH5α with GroESL and Negative Control in 24 hour.
  • Reference
    [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.
Copyright © 2018 iGEM UESTC_China