Team:UESTC-China/Notebook

team

DAYNOTE

  • In March
    The school competition of iGEM in University of Electronic Science and Technology of China was held. After series of individual races and team competitions, excellent people were finally chosen into iGEM team of 2018: UESTC-China. Then we started to demonstrate the feasibility of our project in school competition and searched for some useful and interesting projects.
  • In April
    Through many active discussions and literature review, we decide to degrade the straw. We divided all the members into two groups to explore more useful information. We all actively seek out relevant literatures about metabolic pathways and involved gene to make the ideas more clear.
  • In May
    After four sessions of the group discussion and papers reading, we decided to choose straw degradation as our project. Then we started to carry out primary design of the project and then synthesized some key gene.
  • In June
    We are constantly optimizing our project and verifying the feasibility of the project.
  • In July
    Follow the primary design, we mainly did Molecular cloning experiments this month.
    7.16
    According to the requirements of InterLab，We had transformed E.coli DH5α with these following plasmids :BBa_R0040 、BBa_I20270 、BBa_J364000 、BBa_J364001 、BBa_J364002、BBa_J364007、BBa_J364008、BBa_J3640009.
    7.17
    1. We pick 2 colonies from each of the transformation plates and inoculate in 10 mL LB medium + Chloramphenicol. Grow the cells overnight at 37°C and 220 rpm.
    2. We make three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve.
    7.18
    Cell growth, sampling, and assay.
    7.19
    Because the results did not meet expectations，we repeat the InterLab experiment.
    The transformation of eight plasmids had been completed.
    7.20
    Pick 2 colonies from each of the transformation plates and inoculate in 6 mL LB medium + Chloramphenicol. Grow the cells overnight at 37°C and 220 rpm.
    7.21
    Cell growth, sampling, and assay.
    7.22
    We had constructed some elements for the part updating, which mainly included lacZ ORF fragment, J23100-RBS-pelB-5D, J23100-RBS and J23100-RBS-pelB.
    Designation Primer-F Primer-R Template Size
    lacZ ORF fragment iGEM058-F iGEM058-R iGEM2016-001 3156bp
    J23100-RBS-pelB-5D iGEM059-A iGEM059-DiGEM2016-001 207bp
    J23100-RBS iGEM059-A iGEM059-B iGEM2016-001 124bp
    J23100-RBS-pelB iGEM059-A iGEM059-C iGEM2016-001 192bp
    1.Polymerase Chain Reaction.
    2. Gel Electrophoresis of PCR-Amplified product，and get the target band we expect.
    3.Extraction of DNA from Agarose Gel.
    7.23
    1. USE AXYGEN kit to do plasmid extraction for fragment amplification.
    2. Polymerase Chain Reaction.
    Designation Primer-F Primer-R Size
    part 10 iGEM Y Part10-F iGEM Y Part10-R JSR-08 1243bp
    part 12 iGEM Y Part12-F iGEM Y Part12-R JSR-08 509bp
    part 13 iGEM Y Part13-F iGEM Y Part13-R JSR-09 349bp
    part 17 iGEM Y Part17-F iGEM Y Part17-R JSR-11 835bp
    3.Gel Electrophoresis of PCR-Amplified product，and get the target band we expect.
    4.Extraction of DNA from Agarose Gel.
    5.Restriction Enzyme Digest.
    Designation Enzyme-A Enzyme-B Template
    part 10-cut FD-EcoRI FD-PstI part 10
    part 12-cut FD-EcoRI FD-PstI part 12
    part 13-cut FD-EcoRI FD-PstI part 13
    part 17-cut FD-EcoRI FD-PstI part 17
    PSB1C3-cut FD-EcoRI FD-PstI PSB1C3
    6.Use AXYGEN enzymatic reaction kit to do DNA cleanup.
    7. Mix the four digested fragment with backbone of PSB1C3, then do ligation with T4 ligation enzyme.
    8. Transform the ligation product into E.coli DH5α, Chl-plate.
    7.24
    1.Positive detection of plate part 10, part 12, part 13, part 17. After Gel Electrophoresis, there showed clear band corresponding to the expected sequence size.
    2. Shake bacteria fluid transformed with part 10，part 12，part13，part17 on the shaking table 37℃, 180r, 18h.
    7.25
    Conservation for the bacteria fluid of part 10，part 12， part 13，part 17, then use AXYGEN kit to do extraction.
    7.26
    We have allocated different PH buffers and DNS reagents for enzyme reaction.
    7.27
    1. USE AXYGEN kit to do plasmid extraction for fragment amplification.
    2. Polymerase Chain Reaction.
    Designation Primer-F Primer-R Template Size
    Frag08 Y008-F Y008-R JSR08 3008bp
    Frag09 Y009-F Y009-R JSR09 1531bp
    Frag10 Y010-F Y010-R JSR10 2638bp
    3.Gel Electrophoresis of PCR-Amplified product，and get the target band we expect.
    4. Extraction of DNA from Agarose Gel.
    5. Golden Gate Cloning for piGEM2018-Module003.
    6. Transformed vector piGEM2018-Module003 into E.coli BL21 (DE3).
    7.28
    1. Positive detection of plate piGEM2018-Module003. After Gel Electrophoresis, there showed clear band corresponding to the expected sequence size, 435bp.
    2. Shake bacteria fluid transformed with piGEM2018-Module003 on the shaking table 37℃, 180r, 18h.
    7.29
    1. Conservation for the bacteria fluid of piGEM2018-Module003, then use AXYGEN kit to do extraction.
    2. Restriction Enzyme Digest for verification.
    Designation Enzyme-A Enzyme-B Size
    piGEM2018-Module003 PstI KpnI 7318bp、4248bp
    piGEM2018-Module003 HindIII KpnIII 9696bp、1870bp
    7.30
    1. USE AXYGEN kit to do plasmid extraction for fragment amplification.
    2. Polymerase Chain Reaction.
    Designation Primer-F Primer-R Template Size
    part 11 iGEM Y Part11-F iGEM Y Part11-R JSR-08 1144bp
    part 18 iGEM Y Part18-F iGEM Y Part18-R JSR-11 895bp
    3.Gel Electrophoresis of PCR-Amplified product，and get the target band we expect.
    4. Extraction of DNA from Agarose Gel.
    5. Restriction Enzyme Digest.
    Designation Enzyme-A Enzyme-B Template
    part 11-cut FD-EcoRI FD-PstI part 11
    part 18-cut FD-EcoRI FD-PstI part 18
    PSB1C3-cut FD-EcoRI FD-PstI PSB1C3
    6. Use AXYGEN enzymatic reaction kit to do DNA cleanup.
    7. Mix the four digested fragment with backbone of PSB1C3, then do ligation with T4 ligation enzyme.
    8. Transform the ligation product into E.coli DH5α, Chl-plate.
    7.31
    1. Positive detection of plate part 11, part 18. After Gel Electrophoresis, there showed clear band corresponding to the expected sequence size.
    2.Shake bacteria fluid transformed with part 11，part 18 on the shaking table 37℃, 180r, 18h.
  • In August
    8.1
    Conservation for the bacteria fluid of part 11，part 18, then use AXYGEN kit to do extraction.
    8.3
    Shake bacteria fluid transformed with JSR8 & JSR9 & JSR10 & JSR11on the shaking table 37℃, 180r, 18h.
    8.4
    1. USE AXYGEN kit to do plasmid extraction for fragment amplification.
    2. Polymerase Chain Reaction.
    Designation Primer-F Primer-R Template Size
    part 11 iGEM Y Part11-F iGEM Y Part11-R JSR-08 1144bp
    part 15 iGEM Y Part15-F iGEM Y Part15-R JSR-09 1234bp
    part 16 iGEM Y Part16-F iGEM Y Part16-R JSR-10 2626bp
    part 18 iGEM Y Part18-F iGEM Y Part18-R JSR-11 895bp
    3.Gel Electrophoresis of PCR-Amplified product，and get the target band we expect.
    4. Extraction of DNA from Agarose Gel.
    5. Restriction Enzyme Digest.
    Designation Enzyme-A Enzyme-B Template
    part 11-cut FD-EcoRI FD-PstI part 11
    part 15-cut FD-EcoRI FD-PstI part 15
    part 16-cut FD-EcoRI FD-PstI part 16
    part 18-cut FD-EcoRI FD-PstI part 18
    PSB1C3-cut FD-EcoRI FD-PstI PSB1C3
    6. Use AXYGEN enzymatic reaction kit to do DNA cleanup.
    7. Mix the four digested fragment with backbone of PSB1C3, then do ligation with T4 ligation enzyme.
    8. Transform the ligation product into E.coli DH5α, Chl-plate.
    8.5
    1. Positive detection of plate part 11, part 18. After Gel Electrophoresis, there showed clear band corresponding to the expected sequence size.
    2.Shake bacteria fluid transformed with part 11，part 18 on the shaking table 37℃, 180r, 18h.
    8.6
    Conservation for the bacteria fluid of part 11，part 18, then use AXYGEN kit to do extraction.
    8.7
    1. Shake bacteria fluid transformed with piGEM2018-Module003 on the shaking table 37℃, 180r, 18h.
    2. Shake bacteria fluid transformed with Future001 on the shaking table 37℃, 180r, 18h.
    8.8
    1. Conservation for the bacteria fluid of piGEM2018-Module003, then use AXYGEN kit to do extraction.
    2. Conservation for the bacteria fluid of Future001, then use AXYGEN kit to do extraction.
    3. Restriction Enzyme Digest.
    Designation Enzyme-A Enzyme-B Size
    piGEM2018-Module003 BamHI SpeI 11548bp、18bp
    Future001 BamHI SpeI 2178bp、1812bp
    4. Gel Electrophoresis, and get the target band we expect.
    5. Extraction of DNA from Agarose Gel.
    6. Mix the two digested fragment, then do ligation with T4 ligation enzyme to make piGEM2018-Module004.
    7. Transform the ligation product into E.coli DH5α, Kan-plate.
    8. Preparation of DNS reagent and Acetic acid-Sodium acetate buffer.
    8.9
    1.Positive detection of plate piGEM2018-Module004. After Gel Electrophoresis, there showed clear band corresponding to the expected sequence size, 857bp.
    2.Shake bacteria fluid transformed with piGEM2018-Module004 on the shaking table 37℃, 180r, 18h.
    8.10
    1.Conservation for the bacteria fluid of piGEM2018-Module004, then use AXYGEN kit to do extraction.
    2.Restriction Enzyme Digest for verification.
    Designation Enzyme-A Enzyme-B Size
    piGEM2018-Module004 EcoRI HindIII 7833bp、5893bp
    piGEM2018-Module004 PstI SpeI 8708bp、5018bp
    3.Gel Electrophoresis.
    8.11
    1. USE AXYGEN kit to do plasmid extraction for fragment amplification.
    2. Polymerase Chain Reaction.
    Designation Primer-F Primer-R Template Size
    part 11 iGEM Y Part11-F iGEM Y Part11-R JSR-08 1144bp
    part 18 iGEM Y Part18-F iGEM Y Part18-R JSR-11 895bp
    3.Gel Electrophoresis of PCR-Amplified product，and get the target band we expect.
    4. Extraction of DNA from Agarose Gel.
    5. Restriction Enzyme Digest.
    Designation Enzyme-A Enzyme-B Template
    part 11-cut FD-EcoRI FD-PstI part 11
    part 18-cut FD-EcoRI FD-PstI part 18
    PSB1C3-cut FD-EcoRI FD-PstI PSB1C3
    6. Use AXYGEN enzymatic reaction kit to do DNA cleanup.
    7. Mix the four digested fragment with backbone of PSB1C3, then do ligation with T4 ligation enzyme.
    8. Transform the ligation product into E.coli DH5α, Chl-plate.
    8.12
    1. Positive detection of plate part 11, part 18. After Gel Electrophoresis, there showed clear band corresponding to the expected sequence size.
    2. Shake bacteria fluid transformed with part 11，part 18 on the shaking table 37℃, 180r, 18h.
    8.13
    Conservation for the bacteria fluid of part 11，part 18, then use AXYGEN kit to do extraction.
    8.14
    According to the requirements of InterLab，We had transformed E.coli DH5α with these following plasmids :BBa_R0040 、BBa_I20270 、BBa_J364000 、BBa_J364001 、BBa_J364002、BBa_J364007、BBa_J364008、BBa_J3640009.
    8.15
    1.We pick 2 colonies from each of the transformation plates and inoculate in 10 mL LB medium + Chloramphenicol. Grow the cells overnight at 37°C and 220 rpm.
    2.We make three sets of unit calibration measurements: an OD600 reference point, a particle standard curve, and a fluorescein standard curve.
    8.16
    Cell growth, sampling, and assay.
    8.17
    Because the results did not meet expectations，we repeat the InterLab experiment.
    The transformation of eight plasmids had been completed.
    8.18
    1.Pick 2 colonies from each of the transformation plates and inoculate in 6 mL LB medium
    2.Grow the cells overnight at 37°C and 220 rpm.
    8.19
    1. Cell growth, sampling, and assay.
    2. Shake bacteria fluid transformed with JSR01, JSR02, JSR03 and JSR05 on the shaking table 37℃, 180r, 10h.
    8.20
    1.Conservation for the bacteria fluid of JSR01 & JSR02 & JSR03 & JSR05, then use AXYGEN kit to do extraction.
    2.Golden Gate Cloning for piGEM2018-Module001.
    3.Transform the Golden Gate Cloning product into E.coli DH5α, Amp-plate.
    8.21
    1.Positive detection of plate piGEM2018-Module001. After Gel Electrophoresis, get target bands of piGEM2018-Module001 we expect, 481bp.
    2.Restriction Enzyme Digest for verification.
    Designation Enzyme-A Enzyme-B Size
    piGEM2018-Module001 Ncol EcoR32l 7102bp、3843bp
    piGEM2018-Module001 Ncol Xhol 9315bp、1630bp
    3.Gel Electrophoresis.
    8.22
    1.Use ultrasound to crush cells.
    2.Verify the expression of our design plasmid piGEM2018-Module003 and Future001 in different E. coli strains by SDS-PAGE.
    3.Shake bacteria fluid transformed with JSR8 & JSR9 & JSR10 & JSR11on the shaking table 37℃, 180r, 18h.
    8.24
    1. Transform the Golden Gate Cloning product piGEM2018-Module001 into E.coli BL21 (DE3), Amp-plate.
    2. USE AXYGEN kit to do plasmid extraction for fragment amplification.
    3. Polymerase Chain Reaction.
    Designation Primer-F Primer-R Templation Size
    part 01iGEM Y Part01-F iGEM Y Part01-R JSR-01 2230bp
    part 02 iGEM Y Part02-F iGEM Y Part02-R JSR-02 2635bp
    part 03 iGEM Y Part03-F iGEM Y Part03-R JSR-03 1520bp
    part 05 iGEM Y Part05-F iGEM Y Part05-R JSR-05 1401bp
    part 06 iGEM Y Part06-F iGEM Y Part06-R JSR-06 2128bp
    part 07 iGEM Y Part07-F iGEM Y Part07-R JSR-06 444bp
    part 08 iGEM Y Part08-F iGEM Y Part08-R JSR-07 1681bp
    part 09 iGEM Y Part09-F iGEM Y Part09-R JSR-07 337bp
    part 10iGEM Y Part10-F iGEM Y Part10-R JSR-08 1243bp
    4.Gel Electrophoresis of PCR-Amplified product，and get the target band we expect.
    5. Extraction of DNA from Agarose Gel.
    6. Restriction Enzyme Digest.
    Designation Enzyme-A Enzyme-B Template
    part 01-cut FD-EcoRI FD-PstI part 01
    part 02-cut FD-EcoRI FD-PstI part 02
    part 03-cut FD-EcoRI FD-PstI part 03
    part 05-cut FD-EcoRI FD-PstI part 05
    part 06-cut FD-EcoRI FD-PstI part 06
    part 07-cut FD-EcoRI FD-PstI part 07
    part 08-cut FD-EcoRI FD-PstI part 08
    part 09-cut FD-EcoRI FD-PstI part 09
    part 10-cut FD-EcoRI FD-PstI part 10
    PSB1C3-cut FD-EcoRI FD-PstI PSB1C3
    7. Use AXYGEN enzymatic reaction kit to do DNA cleanup.
    8. Mix the four digested fragment with backbone of PSB1C3, then do ligation with T4 ligation enzyme.
    9. Transform the ligation product into E.coli DH5α, Chl-plate.
    8.25
    1.Positive detection of plate piGEM2018-Module001. After Gel Electrophoresis, get target bands of piGEM2018-Module001 we expect.
    2. LB plates with 0.2% CMC were inoculated with piGEM2018-Module001 & BL21-WT & empty vector control & bought enzyme and incubated at 37℃ for 24h.
    8.26
    1. Congo red assay to detect the degradation of cellulose but get a bad result.
    2. LB plates with 0.2% CMC were inoculated with piGEM2018-Module001 & BL21-WT & empty vector control & positive control commercial enzyme and incubated at 37°C for 24h again.
    8.27
    Congo red assay for celluloses.
    8.29
    The method of gas chromatography was measured because our column can detect butanol. The result showed the production of butanol.
    8.31
    Optical density (A600) measurements of GroESL different overexpression strain with 1% butanol pretreatment. Data are from three biological replicates each with 3 technical replicates.
  • In September
    9.1
    1. We measured the standard curve of butanol by the method of GC.
    2. Use ultrasound to crush cells
    3. Verify the expression of our design plasmid piGEM2018-Module001 in E. coli strains by SDS-PAGE.
    9.2
    1. Improved the heating method and got a better standard curve of glucose.
    2. Prepare the solution of commercialized enzyme and diluted. Then measured absorbance at 540nm to validate if this method works.
    9.3
    1. Shake bacteria fluid transformed with piGEM2018-Module003 on the shaking table 37℃, 180r, 10h.
    2. The overnight culture grown in LB at 37 °C in a rotary shaker (250 rpm) was then inoculated (1% v/v) into fresh TB containing 30 g/L of glucose and kanamycin until OD600=5.
    9.4
    Switched bacteria to anaerobic condition to induce 1-butanol fermentation for 24 hours.
    9.5
    The yield of butanol after 24 hours anaerobic fermentation was measured by GC.
    9.6
    1. Because previous Congo red assay didn't turn out excellent, we had a try to change The ingredients of plates. Added Congo red directly to LB plates instead of Congo red assay. Then inoculated piGEM2018-Module001 and incubated for 24h.But still a bad result.
    2. Incubated piGEM2018-Module001 & BL21-WT & empty vector control with fresh LB media respectively for 48h.
    9.8
    LB plates with 0.2% CMC were inoculated with piGEM2018-Module001 & BL21-WT & Empty vector control & bought enzyme and incubated at 37℃ for 24h again.
    9.9
    1. Use ultrasound to crush cells
    2. Verify the expression of our design plasmid piGEM2018-Module001 in E. coli strains by SDS-PAGE.
    9.13
    Shake bacteria fluid transformed with JSR06 on the shaking table 37℃, 180r, 24h.
    9.14
    1. Use AXYGEN kit to do plasmid extraction for fragment amplification.
    2. Directly convert JSR06 into BL21 (DE3), DH5a, and then culture in medium respectively.
    9.15
    1. Polymerase Chain Reaction.
    Designation Primer-F Primer-R Template Size
    piGEM2018Module003 iGEMPGEL011-F iGEMPGEL011-R JSR-06 2803bp
    2. Gel Electrophoresis of PCR-Amplified product，and get the target band we expect.
    3. Extraction of DNA from Agarose Gel.
    4. Restriction Enzyme Digest.
    Designation Enzyme-A Enzyme-B Template
    piGEM2018Module003-cut FD-NcoⅠ FD-SalⅠ piGEM2018Module003
    5. Use AXYGEN enzymatic reaction kit to do DNA cleanup.
    6. Mix the four digested fragment with backbone（pRQ76）, then do Gibson connection.
    7.Transform the ligation product into E.coli DH5α, Chl-plate.
    9.16
    Double enzyme digestion for piGEM2018Module003.
    Designation
    Designation Enzyme-A Enzyme-B Template
    piGEM2018Module003-cut FD-NcoⅠ FD-EcoRⅠ piGEM2018Module003
    piGEM2018Module003-cut FD-BamHⅠ FD-BglⅡ piGEM2018Module003
    9.17
    1. LB plates with 0.2% CMC were inoculated with piGEM2018-Module001 & empty vector control & positive control enzyme and incubated at 37°C for 24h.
    2. Crushed piGEM2018-Module001 and empty vector by ultrasonic. Incubated them with the solution of cellulose for an hour respectively.
    3.Detected the concentration of glucose but found little variety.
    9.18
    1. Congo red assay for cellulases.
    2. Detected the activity of the cellulase and endo-1,4-β-D-glucanohydrolase through steps above. But we decided to change the buffer. We used Tris-HCl instead of acetic acid-sodium acetate buffer. But still found little variety.
    9.20
    Anylysing results got these days, we found that endo-1,4-β-D-glucanohydrolase is likely to be the rate-limiting enzyme. We considered incubating the mixture for a longer time.
    9.21
    Double enzyme digestion for piGEM2018Module002.
    Designation
    Designation Enzyme-A Enzyme-B Template
    piGEM2018Module003-cut FD-NcoⅠ FD-EcoRⅠ piGEM2018Module003
    piGEM2018Module003-cut FD-BamHⅠ FD-BglⅡ piGEM2018Module003
    9.22
    1. Crushed piGEM2018-Module001 and empty vector by ultrasonic.
    2. Incubated them with the solution of cellulase for seven hours respectively.
    3. Detected the concentration of glucose.
    4. After three groups of repeated experiments, there was a significant difference between piGEM2018-Module001 and empty vector & inactive piGEM2018-Module001 & inactive empty vector.
    However, two of the samples lost. So the data can be unreliable. We need further experiments.
    9.23
    Used ultrasonic to crush piGEM2018-Module001 and empty vector, then stored at 4 ℃.
    9.24
    Incubated them with the solution of cellulase for three hours respectively. Detected the concentration of glucose to calculate the activity of cellulase.
    9.25
    1. Shacked the conservation for the bacteria fluid of piGEM-Module002.
    2. Switched bacteria to anaerobic condition to induce 1-butanol fermentation for 24 hours under different culture conditions.
    3. Measured the titer of butanol by GC.
    9.26
    1. Crushed piGEM2018-Module001 and empty vector by ultrasonic.
    2. Enzyme activity of bifunctional enzymes was measured by DNS method.
    3. There was a significant difference between piGEM2018-Module001 and wild type.
    9.27
    Determination of feruloyl esterase activity of bifunctional enzymes. The absorbance of 0h and 4h under 410nm was determined by using 4-Nitrophenyl trans-ferulate as substrate.
    9.28
    Repeated operations above to detect the activity of the cellulase and endo-1,4-β-D-glucanohydrolase.
    9.29
    Repeated detection of enzyme activity of bifunctional enzymes.
    9.30
    A hydrogen collecting device was built. It is hoped that it can absorb carbon dioxide and obtain pure hydrogen.
  • In October
    10.1
    The standard curve of ferulic acid was determined by gas chromatography.
    10.2
    Ferulic acid can be detected by GC in the supernatant of 12h and 0h fermentation.
    10.3
    Determination of hydrogen standard curve by GC.
    The standard working curve of gas chromatography is based on the single point calibration method of the external standard method, and different amounts of standard gas are injected separately, and the curve is made according to the peak area (peak height).
    10.4
    Verification of high yields of hydrogen.
    Determination of hydrogen production by GC from fermentation bags and anaerobic fermentation equipment.
    At 37 ° C, the glucose concentration was 1% (w/v), and the product was collected by fermentation for 40 hours under the conditions of M9 medium.
    It can be seen that hydrogen production has been significantly improved. ​
    10.10-10.17
    During this time, we mainly resort out the experimental ideas and organize experimental results. Moreover, we improved the wiki various sections, practiced oral English, prepared presentation, made PPT, and performed the rehearsals for many times. We deeply appreciated the joy of team life. We did everything possible to prepare for the final plea, and believed that we would achieve satisfactory grades.
    10.22
    We set out for Boston! Fighting!
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