Week1(4.25-5.1)

Wet

1. Experiment design and check the protocol.

Dry

1. Discuss how to use modeling method in our project and bring up several potential modeling ideas.

HP

1. Human practice program planning and hand out questionnaires.

Week2(5.2-5.8)

Wet

1. Purchase reagents, prepare consumables.
2. Experiment design.

Dry

1. Design physical models in conjunction with enteral drug simulation.

HP

1. Interaction with High School Teenagers.

Week3(5.9-5.15)

Wet

1. Synthesize the target fragments and amplify XylR from Bacillus subtilis.
2. Search for articles containing protocols on the Internet.
3. Prepare each fragment and try overlap extension PCR.
4. Amplify segments: Chem, Temp, EGFP, Holin; extract plasmid: pBE2.

Dry

1. Design the first version device for better enteral drug simulation.

HP

1. Visit professor Yu Yi in college of pharmacy of Wuhan University.

Week4(5.16-5.22)

Wet

1. Use molecular tools like restriction enzyme and ligase to construct recombined plasmid pHT-Sam2 and pBE-PET8; transfer into Escherichia coli DH5α.
2. Try out overlap extension PCR.
3. Digest using restriction enzyme and construct recombined plasmids: pBE2-Chem, pBE2-Temp.
4. Collate the protocols we will use.

HP

1. Communication with DO & GO.

Week5(5.23-5.29)

Wet

1. Identify the positive strains transfered by pHT-sam2 and pBE-XylR and extract plasmids to be sequenced.
2. Redesign protocol based on new vector.
3. Prepare competent cells: E. coli-DH5α; transform recombined plasmids and spread plates.
4. Order enzymes, primers and synthesize fragments.

HP

1. Communication with NJMU-iGEM.

Week6(5.30-6.5)

Wet

1. Identify the positive strains transfered by pBE-XylR and extract plasmids to be sequenced; extract the pHT-Sam2 prepared to transfer Bacillus subtilis.
2. Colony PCR to select positive recombinants for sequencing.
3. Purchase reagents and refine our protocols.

HP

1. Conversation with the freshmen of College of Life sciences in WHU.

Week7(6.6-6.12)

Wet

1. Identify the positive strains transfered by pBE-XylR and extract plasmids to be sequenced; use Two-step Chemical Method to transfer Bacillus subtilis.
2. Colony PCR to select positive recombinants for sequencing.
3. Prepare each fragment and try overlap extension PCR.
4. Prepare experimental consumables such as chemically competent cells.

HP

1. Seek the help of professor Gan Fei from the School of Life Sciences.

Week8(6.13-6.19)

Wet

1. Identify the positive strains transfered by pBE-XylR-PET8 and extract plasmids to be sequenced; use Two-step Chemical Method to transfer Bacillus subtilis.
2. Use the fragment that comes with Gibson Assembly Kit as an control, and do the whole experiment (PCR, gel electrophoresis, gel extraction, Gibson assembly, transformation, colony PCR), to serve as an positive control, to make sure that this whole system is working.
3. Extract plasmids: pBE2-Chem, pBE2-Temp; digest and then construct recombined plasmids: pBE2-Chem-EGFP, pBE2-Temp-EGFP, pBE2-Chem-Holin, pBE2-Temp-Holin.
4. Try out overlap extension PCR.

HP

1. Hold a series of popular science activities with NJMU-iGEM and DO & GO.

Week9(6.20-6.26)

Wet

1. Change the method of coloning pBE-XylR to homologous recombination; use Two-step Chemical Method to transfer Bacillus subtilis.
2. Finish the control experiment and start to do the formal experiment after verification. Obtain and purified linearized fragments and vector, use Gibson Assembly to link the fragment and the vector together and then transform DH5α chemically competent cells.
3. Redesign protocols and separate the construction process into two.
4. Prepare competent cells: E. coli-DH5α; transform recombined plasmids and spread plates.

Dry

1. Purchase parts of enteral drug simulation device and assemble.

HP

1. Communication with DO & GO.

Week10(6.27-7.3)

Wet

1. Use homologous recombination to construct pBE-XylR-PET8; use Two-step Chemical Method to transfer Bacillus subtilis.
2. Link the PCR products again using Gibson Assembly, transform and colony PCR.
3. Colony PCR to select positive recombinants for sequencing.
4. Purchase reagents.

HP

1. Take part in an online meetup.

Week11(7.4-7.10)

Wet

1. Identify the positive strains of pBE-XylR-PET8 and send them to be sequenced; use Two-step Chemical Method to transfer pHT-Sam2 Bacillus subtilis.
2. Order commercial competent cells and prepare our own chemical competent cells once more.
3. Check fragments and construct the first plasmid intermediate.
4. Colony PCR to select positive recombinants for sequencing and preserve bacterial strains.

Week12(7.11-7.17)

Wet

1. Identify the positive strains of pBE-XylR-PET8 and send them to be sequenced; change the method to electrotransformation to transfer pHT-Sam2 Bacillus subtilis.
2. Try the whole experiment again using PCR purification as an alternative way around gel extraction.
3. Construct the final plasmid, colony PCR and sequence to identify.
4. Amplify segments: CinI; extract plasmid: pET-28a.

Dry

1. Design the second version device for better enteral drug simulation.

HP

1. Organize a training for the students in the Elite Program.

Week13(7.18-7.24)

Wet

1. Use electrotransformation to transfer pBE-XylR-PET8 into Bacillus subtilis.
2. Identify the positive Bacillus subtilis of pHT-Sam2 and send them to be sequenced.
3. Analyze the problems and redo the construction.
4. Digest using restriction enzymes and construct recombined plasmids: pET-CinI.

Dry

1. Purchase parts of the second version enteral drug simulation equipment and assemble.

Week14(7.25-7.31)

Wet

1. Explore the induction condition of Sam2 B. subtilis through SDS-PAGE;use electrotransformation to transfer pBE-XylR-PET8 into Bacillus subtilis.
2. Try one more time, from PCR amplificaiton of products to Gibson and colony PCR, using PCR purification rather than gel extraction.
3. Build the variant library and sequence the mutagenesis.
4. Prepare competent cells: E. coli-DH5α, E. coli-BL21; transform recombined plasmids and spread plates.

HP

1. Hold the Central China iGEM Communication.

Week15(8.1-8.7)

Wet

1. Use Western Blot to verify the expression of Sam2; identify the positive strains of pBE-XylR-PET8 and send them to be sequenced.
2. Look up online publications and make modifications to our protocols by assembling multiple fragments for one single time.
3. Build the variant library and sequence the mutagenesis.
4. Colony PCR to select positive recombinants for sequencing.

HP

1. Participate in ICII.

Week16(8.8-8.14)

Wet

1. Explore the induction condition of PET8 B. subtilis through SDS-PAGE
2. Use Western Blot to verify the expression of pHT-Sam2; identify the positive strains of pBE-XylR-PET8 and send them to be sequenced.
3. Separate the desired variant through selection culture.
4. Extract plasmids: pET-CinI; transform recombined plasmids and spread plates.

Dry

1. Design portable dual port filter.

HP

1. Participate in CCiC.

Week17(8.15-8.21)

Wet

1. Use Western Blot to verify the expression of pHT-Sam2; identify the positive strains of PET8 and send them to be sequenced.
2. Do another round of molecular cloning and amplify the fragments that we will ligate together; design new primers.
3. Separate the desired variant through selection culture.
4. Colony PCR to select positive recombinant for sequencing; preserve bacterial strains.

Dry

1. Purchase parts of portable dual port filter and assemble.

HP

1. iGEM introduction for 2022 freshmen.

Week18(8.22-8.28)

Wet

1. Purchase new antibodies and repeat the Western Blot to verify the expression of Sam2 and PET8.
2. Amplify the fragments to be ligated again successfully and do PCR amplification and Gibson Assembly.
3. New cycles of selection and sequencing identification.
4. Transform plasmids: pBE2-Chem-Holin, pBE2-Temp-Holin into B. subtilis and spread plates.

HP

1. Investigate and survey in MGI.

Week19(8.29-9.4)

Wet

1. Repeat Western Blot to verify the expression of Sam2 and PET8;
2. Use fluorescence microscopy to verify the localizaiton of PET8 through its fused sfGFP.
3. Colony PCR to select positive recombinants for sequencing and construct the intact repressilator in E. coli and B. subtilis.
4. New cycles of selection and sequencing identification.

Dry

1. Complete enteral drug simulation experiment.

Week20(9.5-9.11)

Wet

1. Use fluorescence microscopy to verify the localizaiton of PET8 through its fused sfGFP.
2. Design colony PCR primers for the detection of co-transformation; extract the Repressilator plasmid and the reporter plasmid; prepare for the competent cells using E. coli that already contains one of the plasmids.
3. Develop the procedures and conduct a new round of selection.
4. Colony PCR to select positive recombinants for sequencing; preserve bacterial strains.

HP

1. Hand out questionnaires.

Week21(9.13-9.18)

Wet

1. Identify the positive strains of pBE-XylR-PET8 and send them to be sequenced; change the method to electrotransformation to transfer pHT-Sam2 Bacillus subtilis.
2. Try the whole experiment again using PCR purification as an alternative way around gel extraction.
3. Construct the final plasmid, colony PCR and sequence to identify.
4. Amplify segments: CinI; extract plasmid: pET-28a.

Dry

1. Complete the test experiment of the portable dual-port filter.

HP

1. Participate in ICII.

Week22(9.19-9.25)

Wet

1. According to the result, redesign the biobricks about Sam2 and PET8p.
2. Using the co-transformed strain, we test the fluorescence under microplate reader and under the fluorescence microscope.
3. Develop the procedures and conduct a new round of selection.
4. Quantify fluorescence in E. coli and concentration of B. subtilis to verify.

Dry

1. Revise our hardware articles and translate them into English.