Team:WHU-China/Notebook

Wet Lab > Notebook
Notebook

Wet

  1. We tested the condition of inducing the GFP to express.

  2. Experiment preparation and extraction of pET26b+.

  3. Search the literature and identify the target gene for knockout experiment.

  4. Obtained relevant gene sequence information and designed expression vector.

HP

  1. Interview professors in related fields, introduce our project to them and ask them to make suggestions to help us refine the design and improve the feasibility of the project.

  2. In conjunction with world skin care days, skin health-related tweets are tweeted within our official account.

Wet

  1. We tested the condition of inducing the GFP to express, and asked NTHU for advice about how to diffuse the oleic acid.

  2. Extraction of vector pUC-PctA and construction of vector pET26b+-PctA.

  3. Contact the professor's lab for material support and technical guidance.Experiment preparation.

  4. Designed and synthesized the primer of fadD and fadE.

HP

  1. Etodole surrounding community, to popularize the knowledge of skin health as well as acne for the group of elementary school students.

Wet

  1. We devoted to find out the suitable way measuring the fluorescence intensity and make it.

  2. First attempt to transform pET26b+-PctA into E. coli Dh5alpha.

  3. Conduct the first round of hisBCD gene knockout.

  4. Construction of the recombinant vectors of FadD and FadE.

HP

  1. Participation in the CCiC HP discussion meeting.

Wet

  1. We explore how the concentration of oleic acid inluence the express of GFP.

  2. Extraction and PCR identification of pET26b+ and pUC67.

  3. Conduct the second round of hisBCD gene knockout.

  4. Construction of the recombinant vectors of FadD and FadE.

Wet

  1. We explore how the time of induction influence the express of GFP.

  2. Second attempt to transform pET26b+-PctA into E. coli Dh5alpha.

  3. Conduct the second round of hisBCD gene knockout4.

  4. Construction of the recombinant vectors of FadD and FadE.

Dry

  1. Discussed how to use modeling method in our project and brought up several potential modeling ideas

HP

  1. Exchange the progress and experience of model establishment with HUST2-China.

Wet

  1. Searched for the articles that concerned FadR and directed evolution on the internet;

  2. Extraction of pET26b+ and pUC67

  3. Analyze the reasons for the experiment failure. Search the literature and preliminarily determine the feasibility of using CRISPR-Cas9 as gene editing tools in E. coli.

  4. Try to explore the best expression conditions of the target protein(FadD,FadE)

Dry

  1. Designed physical models in conjunction with directed evolution projects

HP

  1. Contact relevant pharmaceutical enterprises to prepare for the commercialization of the project.

Wet

  1. Reorganized all the mathods used in the articles we have read, and start searching for the advantages and disadvantages of each method;

  2. Extraction of pET26b+ and pUC67 and enzyme cut.

  3. Search the literature and choose glyA as target knockout.

  4. Try to explore the best expression conditions of the target protein(FadD,FadE)

Dry

  1. Determined to use Microfluididic chips to build a high throughput directional evolution platform

HP

  1. Participate in the "Luojiazhihang" activity organized by Wuhan University and practice for the commercialization and real world application of our project.

Wet

  1. Through discussion, decided on the method best suited for our project, and started searching for protocols in the internet and prepare for the experiment;

  2. Construction of pET26b+-PctA.

  3. Conduct the first round of glyA gene knockout based on DH5α strain.

  4. Try to explore the best expression conditions of the target protein(FadD,FadE)

Dry

  1. Used L-edit to design chip structure

Wet

  1. Designed plasmid and primers, and ordered on the internet.

  2. Conduct the second round of glyA gene knockout based on DH5α strain.

  3. Induced protein expression and detected the ability of engineered bacteria β- oxidative metabolism.

Dry

  1. Lithographic printed mask and first edition chip

HP

  1. Participate in iGEM central China exchange meeting and carry out project exchange and cooperation with iGEM teams in Central China.

Wet

  1. Tried different conditions of epPCR, in search for the best condition.

  2. Successful identification of pET26b+-PctA in DH5alpha.

  3. Conduct the second round of glyA gene knockout based on DH5α strain and analyse the sequencing results. There may be something wrong with the primers, so we redesigned the primers and decided to try again.We also chose MG1655 as a new chassis for gene knockout experiment in case of the influence of DH5α itself.

  4. Induced protein expression and detected the ability of engineered bacteria β- oxidative metabolism.

Dry

  1. Pre-tested the first version of the chip

HP

  1. Cooperate with the high school team (XHD-Wuhan-Pro), help them improve their project and visit the laboratory.

Wet

  1. Extracted genomic DNA, and used the genomic DNA as the template of PCR and epPCR to obtained FadR.

  2. Extraction of pET26b+-PctA from DH5alpha and transformation of it into E. coli BL21(DE3)

  3. Contact with the professor's lab to attain MG1655 strain.Extract pKD46 and pKD13.

  4. Purified the target protein with nickel column and then detected its expression with SDS-PAGE electrophoresis.

Dry

  1. Constructed growth simulator model to predict the coculture of engineering bacteria and P. acnes by differential equations.

  2. Redesigned the second version of the chip

Wet

  1. Performed PCR product purification, enzyme cut, gel electrophoresis and gel purification of FadR, but found that the purified product has such a low concentration that it is hard to continue in the next step.

  2. Failed identification of pET-26b+-PctA in BL21(DE3) and second transformation of pET26b+-PctA.

  3. We extracted the pKD13 plasmid from the lab-supplied strain, but encountered some problems in culturing the strain, which did not seem to grow on kanamycin resistant plates. In the process of communication with the laboratory,we learned that the graduate students were also troubled by this problem, but the dilemma is that we couldn't obtain plasmids from other sources for the time being.

Dry

  1. Used simulated data to solve the parameters in growth simulator and made sure the momdel was usable.

  2. Brought up the FBA model.

  3. Remade the second version chip and tested the performance

HP

  1. Cooperate with CAU-China and HUST-China to conduct online publicity and complete public education.

  2. Interview Dr. Min Wang, a plastic surgeon of Central South Hospital, to understand the cutting-edge acne treatment strategies.

Wet

  1. Changed some experimental conditions and tried again to get FadR digested with enzyme.

  2. Successful sequencing for pET26b+-PctA in BL21(DE3) and SDS-PAGE for E. coli BL21(DE3) pET26b+-PctA

  3. Culture strains containing pKD13 from previous lab and contact with other labs for pKD13.

Dry

  1. Made beads loading,bacteria loading,and water in oil droplets in the second version of the chip

  2. Found data and solved the FBA model

HP

  1. Interview Prof. Zhixiongxie and ask for the problems encountered in the experiment for a second visit.

Wet

  1. Tried to get FadR digested with enzyme again, this time using E. coli DH5α as a template directly, without the step of genomic DNA extraction, and tried new gel extration kit, which yield a better rate of recovery.

  2. SDS-PAGE orthogonal experiment of induced expression of pET26b+-PctA and optimization of experiment conditions

Dry

  1. Searched online FBA tools

HP

  1. Participate in CCiC activities.

  2. Interview Renfu pharmaceutical and other related pharmaceutical enterprises.

Wet

  1. Tried to get pBAD33 digested with enzyme, joined DNA fragments and transformed into DH5α., when the product was linked into the plasmid, no transformant were found.

  2. SDS-PAGE orthogonal experiment of induced expression of pET26b+-PctA and optimization of experiment conditions

  3. Conduct the third round of glyA gene knockout based on MG1655 strain. 4. Access to information and analyze the reason why the molecular weight of protein is lower than expected.

Dry

  1. Observed the capture efficiency was observed under a microscope

  2. Used Escher tool to build up a new FBA model

HP

  1. Interview the counters of skin care products to get to know the sales of skin care products.

  2. Carry out science popularition on synthetic biology and acne treatment for students in the middle school attached to Wuhan University.

Wet

  1. Performed epPCR and PCR again using the bacteria as the template to get FadR for many times, but after electrophoresis, there are only diffusion band.

  2. Optimization of experimental conditions of SDS-PAGE and induction of pET-26b+-PctA.

  3. Conduct the third round of glyA gene knockout based on MG1655 strain.

  4. Changed the engineering strain - the E. coli DH5α strain was replaced with BL21 strain.

Dry

  1. Designed and printed in 3-D for microscope viewing

  2. Assigned tasks to sort out modeling results and write articles

HP

  1. Interview Dr. Ding Hong, a professor in the field of acne treatment again, and ask about what kind of product form is more suitable for our project.

Wet

  1. Wondered whether the diffusion band is due to degration of the primer or due to contemination of the PCR mastermix, another mastermix and another primer were ordered.

  2. Salt fractionation purification of PctA and SDS-PAGE of unpurified and purified PctA

  3. Conduct the fourth round of glyA gene knockout based on DH5α strain.

  4. Induced the target protein in E. coli BL21 strain and then purified the target protein with nickel colum,detected its expression with SDS-PAGE electrophoresis

HP

  1. Participate in the project defense of "Luojiazhihang" and successfully conclude the project.

  2. Popularize synthetic biology and iGEM to undergraduates in the college of life sciences of Wuhan University.

Wet

  1. Performed both kinds of PCR again, using the new agents we have ordered, still using the genomic DNA as the template, this time, there are correspounding band after the gel electrophoresis.

  2. Ni-IMAC of PctA and SDS-PAGE of unpurified and purified PctA.

  3. Conduct the fourth round of glyA gene knockout based on DH5α strain. Design the primers and sequence genes. Analyse the data.

Dry

  1. Designed the third version chip for better performance in water in oil droplets

Wet

  1. Due to the lack of time, the product wasn't linked into the plasmid, but rather, it was sequenced to see if there were indeed a higher mutation rate after the epPCR, compared to the normal PCR.

  2. Western blot of unpurified and purified PctA and design of pET26b+-OmpA-PctA with optimized signal sequence

Dry

  1. Revised our modeling articles and translated them into English.

HP

  1. Participate in ICII and publicize iGEM for the whole school.

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