11.13
Instructor’s speech
The brief introduction of the team members.
Classification into five groups, including wet lab, dry lab, human practice, network, and design.
Safety training
Our advisor trained us on experimental safety, including proper use of the instrument, proper wear in the lab,
and other considerations.
Get access to necessary articles to have a full understanding of the project; Summarize the difficulty of hydroxylating steroid substance via chemical synthesis;
11.21
-
Experiment preparation:
(1) Configure the TB mediumIntegrant Dosage Tryptone 12 g/L Glycerinum 5 g/L Yeast extract 24 g/L KH2PO4 17 mmol/L K2HPO4 72 mmol/L 121℃ sterilization for 15 min (2) Configure the LB medium
Integrant Dosage/th> Tryptone 10 g/L Yeast extract 5 g/L NaCl 10 g/L 121℃ sterilization for 15 min 11.25
- (1) Clean reagent bottles
- (2) Insert pipette tips
Review of literature related to hydroxylation of steroids; Turn to biosynthesis for help and discover an appropriate enzyme that is important to hydroxylate the site we want. Decide to use CYP107D1 (Olep) to hydroxylate steroids.
11.27
Construction of the strain E. coli O1.
12.1
Full wavelength scanning of Olep
12.4
SDS-PAGE analysis of crude enzymes, intracellular P450 enzymes, intracellular and extracellular P450 enzymes
12.8
Explore TLC methods for detecting DCA and 6β-OH DCA
HPLC test product content
TLC results for DCA and 6β-OH DCA
Increase the soluble expression of Olep
12.12-12.21: Screening of different plasmid vectors
12.12
- Construct plasmids:
- pETDuet-camA-camB-oleP;
- pACYCDuet-camA-camB-oleP;
- pRSFDuet-camA-camB-oleP.
12.14
- Plasmid extraction
- The constructed plasmid was sent to the company for sequencing.
12.18
- Whole-cell-catalyzed reactions(based on final concentration): Strain cell OD600=30, deoxycholic acid 1 mg/mL, NADPH 1 mM, glucose dehydrogenase 1 U/mL.
12.20
- TLC test product content
- HPLC test product content
12.22-27: Screening of host cells
12.22
- Transfection plasmid pRSFDuet-camA-camB-oleP into different host cells
12.26
- TLC methods detect DCA and 6β-OH DCA
12.28 – 1.7：Screening for pro-soluble labels
12.29
- Construct plasmids: PCR
- SUMO-linker-Olep
- MBP-linker-Olep
- TF-linker-Olep
- Trx-linker-Olep
- Nus-linker-Olep
- GST-linker-Olep
12.31
- The constructed plasmid was sent to the company for sequencing.
1.2
- Some strains didn't grow very well, so the agar streak culture was re-performed. For those strains that grow well, we selected some strains with complete shapes on each plate to be sent for sequencing.
1.4
- The strains were still not growing very well. We suspect this may be because the receptive cells have been preserved for too long. So we reprepared the receptive cells.
1.8
- The growth of the strain was significantly improved compared with that before
1.11
- Whole-cell-catalyzed reactions
1.13-1.18: Optimization of culture conditions
1.15
- Transfer to shaker and culture, then induce. When inducing, we culture the cells respectively at 16 °C, 20 °C, 25 °C, and 30 °C. We control the inducer(IPTG) concentration at 0.1mmol/L, 0.25mmol/L, 0.5 mmol/L, 0.75 mmol/L, and 1.0 mmol/L, respectively
1.17
- TLC test product content
Spring Festival (Chinese New Year)!
Increase intracellular heme supply
1.30-2.5：Measurement of heme binding efficiency of MBP and TF
1.30
- Agar streak: The previously preserved MBP-oleP and TF-oleP recombinant strains were removed and marked on LB medium for activation Cultured for 12h, then inoculated into liquid LB medium
2.2
- Whole-cell-catalyzed reactions
2.4
- Measured the heme concentration by UHPLC-QTOF-MS
2.6-2.12：Exogenous addition of heme and its precursor
2.8
- During induction, added 0.025 mL of inducer isopropyl-β-d-thiogalactoside (1 M IPTG, final concentration 0.5 mM). Then added 5-amino-ketovaleric acid (ALA)＋FeSO4 ，5-amino-ketovaleric acid (ALA)＋FeCl3, heme, respectively. Then the culture was continued at 25℃ for 24-32 h.
2.11
- TLC detected product content;
- HPLC detected product content;
2.13-2.25: Enhancing heme biosynthetic pathway
2.14
- Construct plasmids: PCR
- pCDFDuet-hemA-hemL;
- pETDuet-hemB-hemC-hemD-hemH;
2.20
- Transfer to shaker and culture, then induce.
2.23
- Whole-cell-catalyzed reactions
2.24
- TLC test product generation
Screening and modification of redox partners
Week 17 - Week 19: Redox partner Sensor Construction
3.15
- Construct plasmids: PCR
- pRSFDuet-BM3-GFP-1-10-GFP-11-olep;
- pRSFDuet-camA-camB-GFP-1-10-GFP-11-olep;
- pRSFDuet-FdR0978- Fdx1499-GFP-1-10-GFP-11-olep;
- pRSFDuet-petH-petF-GFP-1-10-GFP-11-olep.
3.18
- Plasmid extraction, send them to the company for sequencing
3.22
- After the fermentation was completed, add 200 uL of the bacterium to a 96-well plate to determine the biomass (wavelength 600 nm) and fluorescence value (excitation wavelength 488 nm, emission wavelength 520 nm) using an enzyme labeler.
Week 20 - Week 22: Verifying the reliability of the redox partner sensor
3.25
- Construct plasmids: PCR
- pRSFDuet-BM3-olep;
- pRSFDuet-camA-camB-olep;
- pRSFDuet-FdR0978-Fdx1499-olep;
- pRSFDuet-petH-petF-olep.
3.26
- The target gene fragment is attached to the plasmid vector and then transferred into the host cell
3.29
- One plasmid (pRSFDuet-FdR0978-Fdx1499-olep) failed to build, rebuild
4.3
- Transfer to shaker and culture, then induce.
4.7
- HPLC test product content
Week 23 - Week 25: Redox partner fusion assortment
4.11
- Construct plasmids: PCR
- recombinant strains R6-R9
4.16
- Whole-cell-catalyzed reactions
Construct the optimal whole-cell catalytic system
Targeted mutate key sites on ferredoxin petF
6.20
- Construct plasmids: PCR
Bands were detected by DNA gel electrophoresis
6.22
- The target gene fragment is attached to the plasmid vector and then transferred into the host cell
6.25
- The sequencing results of some plasmids were incorrect, so we tried different methods to attach DNA fragments to plasmids.
7.1
- The sequencing results of some plasmids were incorrect. This time we redesigned the primers and did PCR again.
7.6
- The sequencing results were correct. Agar streak, cultured 12 h
7.8
- Bacteria with complete shape were selected for sequencing.
7.9
- The sequencing results of some plasmids were incorrect. So we selected several strains from the original culture medium and sent them to the company for sequencing again.
7.10
- The sequencing results of some plasmids were still incorrect. So we repeated the agar streak.
7.11
- The chromogenic agent used for TLC is used up. Reconfigure.
| Integrant | Dosage |
|---|---|
| H2O | 225 mL |
| Ammonium molybdate tetrahydrate | 12.5 g |
| Cerous molybdate | 2.5 g |
| H2SO4 | 25 mL |
7.13
- The sequencing results of several strains were incorrect. However, the sequence comparison results showed that only a few nucleotides were inconsistent, so we selected several strains from the original plate and sent them with the previous strains for sequencing.
7.15
- It turned out that the previous sequencing results were wrong. All strains had been constructed, and the agar streak began.
7.17
- Transfer to shaker and culture, then induce.
7.20
- After the fermentation was completed, 200uL of the bacterium was added to a 96-well plate to determine the biomass (wavelength 600 nm) and fluorescence value (excitation wavelength 488 nm, emission wavelength 520 nm) using an enzyme labeler.
7.30
- We counted all the fluorescence intensities, and selected the recombinant strains with strong fluorescence intensity. The primers were redesigned for catalysis.
8.3
- Construct plasmids: PCR
8.5
- The target gene fragment is attached to the plasmid vector and then transferred into the host cell. This time we took the previous lesson and used a variety of enzymes to link.
8.7
- The sequencing results were incorrect, so we attached DNA fragments to plasmids again.
8.10
- The sequencing results were correct. Agar streak, cultured 12h.
8.12
- We selected some strains with complete shapes on each plate to be sent for sequencing.
8.14
- The sequencing results of several strains were incorrect, so we selected several strains from the original plate and sent them with the previous strains for sequencing. At the same time, we also attached DNA fragments to plasmids again.
8.17
- The sequencing results of the reconstructed plasmids were satisfactory. Agar streak, cultured 12h.
8.20
- The results of strain sequencing were correct, all strains had been constructed, and the agar streak began.
8.21
- Transfer to shaker culture.
9 mutants + wild type + negative control were fermented in 50 ml/250 ml system
8.23
- Whole-cell-catalyzed reactions.
8.24
- TLC test product content 200 uL methanol redissolved sample was centrifuged at 14000 rpm for 20 min, for the liquid phase and TLC
8.26
- HPLC test product content
