Calendar

March

1. Determined the project, had an overview of project, and divided the project into three parts:

Part 1. Express the cellulases in the Zymomonas mobilis

Part 2. Build a metabolic pathway to produce bioplastic of poly-β-hydroxybutyrate (PHB) in Z. mobilis

Part 3. Build a related model

Part 4. Human practices

2. Selected the required gene of different cellulases by investigating available literature.

3. Wrote a preliminary experimental design.

April

Part 1.

1. Consulted the literature to obtain information with cellulase and identified three enzymes: exoglucanase (CBH ), endoglucanase (EG ) and Beta glucanase (BGL ).

2. Compared the activity of these enzymes through literature and selected eleven cellulases from different strains.

3. Searched the EC number or accession number of the enzyme related information form KEGG (https://www.kegg.jp/). Identified the genes fragments from NCBI database.

Promoter: Pgap, Peno, Peda, Ptet

Signal peptide: phoC, phoD, 0331, GFOR

Terminator: T0152r, T1779r, T1929f

Part 2.

1. Searched the literature of PHB production, and studied its research method and application .

2. Identified the source strain of the genes related to PHB production, Ralstonia eutropha, and designed the metabolic pathway of PHB production in Z. mobilis.

3. Designed experiment.

Part 3.

1. Decided the research direction of model.

2. Studied the mechanism of transcriptional termination.

Part 4.

1. Organized a team for Human Practices.

May

Part 1.

1. Set up a series of plasmid maps and optimized them.

2. Built pEZ15A-Pgap-CelA-T0152r plasmid, and used for signal peptide replacement.

3. Received the plasmids from the research team of Yuanyuan Ma, Tianjin University

①pEZ15A-Pgap-GFOR-SfBGL-T0117f

②pEZ15A-Ptet-PhoD-RuBGL-T0117f

4. These two plasmids are used by us to transfer into e. coli to express cellulases.

Part 2.

1. Selected the tetracycline-inducible promoter Ptet for construction of plasmid.

2. Used Gibson assembly method to construct plasmids pEZ15A-Ptet-phbCAB.

Part 3.

1. Measured terminator strength by using dual fluorescence report system.

2. Found sequence character of terminator.

Part 4.

1. Conducted a presentation in school to introduce what is iGEM . Determined the details of ‘recycling of cartons’ investigationt.

June

Part 1.

1. Replaced the signal peptide with 0331, PhoC, PhoD, GFOR based on the plasmid pEZ15A-Pgap-CelA-T0152r by using PCR, overlapping and one-step cloningto construct the following plasmids:

① pEZ15A-Pgap-CelA-T0152r

② pEZ15A-Pgap-PhoD-CelA-T0152r

③ pEZ15A-Pgap-0331-CelA-T0152r

④ pEZ15A-Pgap-GFOR-CelA-T0152r

⑤ pEZ15A-Pgap-PhoC-CelA-T0152r

⑥ pEZ15A-Peno-PhoC-BGLA-T0117f

2. Used the 3,5-dinitrosalicylic acid (DNS) method to measure the enzyme activities.

Part 2.

1. Selected High performance liquid chromatography (HPLC) to detect PHB content.

2. Transferred the pEZ15A-Ptet-phbCAB plasmid into Z. mobilis.

3. Set different tetracycline concentration gradient to induce PHB accumulation in Z. mobilis.

4. Made growth curve, glucose consumption curve, ethanol production map, and dry weight map.

Part 3.

1. Found how to predict secondary structure of terminator.

2. Studied about the crystal structure of RNA polymerase (RANP) in different states.

Part 4.

1. Submitted a human practice plan to the school and got some help.

July

Part 1.

1. Because of the DNS reagent allows the simple and rapid quantification of reducing sugars, we determined to use DNS to come out the enzyme activities.

2. Obtained plasmids and recombinant strains of Z. mobilis:

① pEZ15A-Pgap-CelA-T0152r

② pEZ15A-Pgap-0331-CelA-T0152r

③ pEZ15A-Pgap-PhoC-CelA-T0152r

④ pEZ15A-Pgap-PhoD-CelA-T0152r

⑤ pEZ15A-Pgap-GFOR-CelA-T0152r

3. Measured the enzyme activity of EG.

4. Optimized our scheme of the enzyme activity measurement though studied a doctoral thesis on the enzyme activity determination of Z. mobilis cellulose named Secretive Expression of Cellulase Enzyme and Its Application in Biorefinery Processes from China national knowledge infrastructure.

5. Used the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to verify the expression of cellulases.

6. Detected the generation of enzymeswith Western blot .

7. Determined the method of using Flag-tag to verify protein expression.

Part 2.

1. Used HPLC to measure PHB production. Analyzed the results of the basic fermentation and concluded that the recombinant Z. mobilis could produce PHB, but the production was not high.

2. Designed methods to increase the production of PHB.

3. Optimized the metabolic pathway for PHB production:

1) Increased the content of NADPH:

① Enhanced the expression of the glucose-6-phosphate dehydrogenase (ZMO0367) pEZ15A-Pgap-phbCAB.

② Enhanced the expression of NAD+ kinase (ZMO1329).

2) Increased the precursor content of acetyl-CoA:

① Enhanced the expression of dihydrothiocinamide acetyl transferase (ZMO0510) pEZ15A-Pgap-phbCAB.

② Enhanced the expression of dihydrothiocinamide dehydrogenase (ZMO0512).

4. Selected biological components such as strong promoter Pgap and designed the plasmid mapping.

Part 3.

1. Looked for nucleic acid intermolecular force parameters.

2. Studied the criteria for selecting evaluation parameters.

3. Determined the parameters used for prediction and the method of sequence feature recognition.

Part 4.

1. Prepared the materials needed for the investigation and contacted the relevant person in charge.

August

Part 1.

1. Further modified the methods to measure enzyme activities.

2. Used PCR、Overlap extension PCR and One-step cloning to construct plasmids.

① pEZ15A-Pgap-CelA-Flag-T0152r

② pEZ15A-Peda-0331-ExgS-Flag-T1751f

③ pEZ15A-Peda-0331-Cel9K-Flag-T1751f

④ pEZ15A-Peno-PhoC-BGLA-Flag-T0117f

⑤ pEZ15A-Pgap-PhoD-AC E1-2-Flag-T0152r

⑥ pEZ15A-Pgap-PhoD-EG EG-2-Flag-T0152r

⑦ pEZ15A-Peda-0331-Cbh9A-Flag-T1751f

Part 2.

1. Used Gibson assembly, overlap extension PCR and One-step cloning to construct plasmids.

① pEZ15A-Pgap-phbCAB

② pEZ15A-Pgap-ZMO0367-phbCAB

③ pEZ15A-Pgap-ZMO1329-phbCAB

④ pEZ15A-Pgap-ZMO0510-phbCAB

⑤ pEZ15A-Pgap-ZMO0512-phbCAB

Part 3.

1. Studied the program to calculate the free energy for terminator.

2. Wrote python scrips to build a workflow for structure prediction and energy calculation.

3. Built a workflow to find sequence character.

Part 4.

Completed social surveys and analyzed data.

1. Distributed 100 questionnaires to residents in Xujiapeng residential area of Wuhan on how to deal with used courier boxes.

2. Visited the Hubei University STO. Expressed delivery point and the rookie station to learn about the two directions of the express carton: re-use as a courier box or be paid for in the form of waste.

3. Went to the Central China Supervision Authority of the National Energy Administration (NEA) to learn which new energy needs to be developed.

4. Went to the ‘Yaodong Resources’ Plastics Recycling Company in Jiangxia District for on-the-spot investigation.

5. Visited the enterprise workshop in Wuhan ‘Huali biomaterial’ company, the largest manufacturer of environmentally friendly packaging products for biomaterials.

6. Two members of HUBU-WUHAN team participated in the 6th Community of China iGEMer Community (CCiC) held in Shenzhen to report the project results and to display the posters.

7. At the end of August, we communicated with iGEM teams from four universities in central China about the experimental progress and problems. We got a lot of suggestions for improvement.

8. Determined the cooperation with the team of iGEM, CAU_China, ECUST_China, XMU-China .

September

Part 1.

1. Tried to import Cel9D into the genome using CRISPR-Cas9 technology.

Part 2.

1. Transferred the pEZ15A-Ptet-phbCAB into Z. mobilis and conducted fermentation experiments many times.

2. Measured the OD_600nm, glucose and ethanol content during fermentation, and made growth, glucose consumption and ethanol production curve .

3. Made standard curve for PHB measurement by HPLC method.

4. Measured PHB production by HPLC.

Part 3.

1. Perfected the work flow of terminator secondary structure prediction.

2. Tested work flow.

Part 4.

1. Conducted a speech to introduce what is iGEM to freshman in Hubei university. Completed the survey of ‘The degree of teachers and students of the school know about iGEM ’and analyzed the data.

October

Part 1.

1. Started a new part to explore the effect of six bases after Ptet promoter.

1.1 Constructed the plasmids of ZM4-pEZ-Ptet-mCherry and ZM4-pEZ-Ptet-W-mCherry.

1.2 Transferred the plasmids into Z. mobilis and conducted fermentation experiments many times.

1.3 Detected the fluorescence by flow cytometry and analyzed the experimental result.

2. Propagated Cel9D to discard the imported plasmid and verified it.

3. Continued to constructe the unfinished plasmids.

Part 2.

1. Integrated Pgap-ZMO0367-phbCAB genes into the Z. mobilis genome.

2. Used the recombinant strain to made a fermentation in cellulolytic solution.

Part 3.

1. Collated and studied the medal standards.

2. Perfected whole workflow with a big dataset.

Part 4.

1. Went to Caidian district No. 6 primary school to carry out an interesting lecture on biology.

Team:HUBU-WUHAN/Calendar

Calendar

March

1. Determined the project, had an overview of project, and divided the project into three parts:

2. Selected the required gene of different cellulases by investigating available literature.

3. Wrote a preliminary experimental design.

April

Part 1.

Part 2.

Part 3.

Part 4.

May

Part 1.

Part 2.

Part 3.

Part 4.

June

Part 1.

Part 2.

Part 3.

Part 4.

July

Part 1.

Part 2.

Part 3.

Part 4.

August

Part 1.

Part 2.

Part 3.

Part 4.

September

Part 1.

Part 2.

Part 3.

Part 4.

October

Part 1.

Part 2.

Part 3.

Part 4.