. Notebook .

1. Wetlab

March 2024

1. We learned the basic theory of synthetic biology and the experimental scheme of strain modification.
2. We received basic experimental skills training such as bacterial culture, plasmid construction, SDS-PAGE and Western blot.
3. Through literature review and discussion, we studied how to improve the targeting and adaptability of OMVs to adapt to different application scenarios.
4. We learned from the previous team's work in ensuring safety in the project and learned from it to better manage the safety management issues in the project.

April 2024

1. We conducted pre-experiments to explore the suitable conditions for preparing protoplasts using liposome extruders.
2. Based on previous work in the laboratory, we constructed self-assembled plasmids of Cas9 RNP and transferred them to EcN for expression (Fig.1).


Fig.1 Schematic diagram of Cas9 RNP synthesis process by E. coli biological self-assembly.

3. We have found a suitable membrane surface display element from the literature. We plan to use Im7/CL7 orthogonal protein pairs to display Im7 protein on the surface of EcN membrane. Then use the CL7 protein that fuses with the antibody co-incubate with OMVs to realize the display of a variety of targeted antibodies.

May 2024

1. The experimental results showed that the use of tac promoters to transcribe gRNA and assemble Cas9 RNP was not efficient in EcN expression and assembly. Therefore, we decided to replace it with a strong promoter to improve the assembly efficiency.
2. A literature review found that wild EcN lacks T7 RNA polymerase that recognizes T7 strong promoters. To this end, we designed an experiment to knock T7 RNA polymerase gene into the EcN genome to enhance gRNA transcription. Subsequently, we prepared OMVs using a liposome extruder and characterized them by transmission electron microscopy (Fig.2).


Fig.2 TEM image analysis and DLS analysis results of OMVs

3. We began to query the sequences of Im7/CL7 proteins in NCBI, and constructed plasmids displaying Im7 proteins on the membrane surface and CL7-fused sfGFP plasmids in SnapGene (Fig.3).


Fig.3 Plasmid profile of Im7 and CL7-sfGFP expression

June 2024

1. We successfully modified the EcN strain and transferred to the Cas9 RNP self-assembly plasmid with replaced promoter for expression. Subsequently, we purified the protein and performed in vitro activity validation experiments to test the efficiency of Cas9 RNP in cutting target plasmids.
2. We transferred the constructed pCold-Inak-Im7 plasmid into EcN and pET23a-CL7-sfGFP into Escherichia coli BL21. The success of EcN expression of Im7 protein and BL21 expression of CL7-sfGFP was verified, and protoplasts of engineered EcN were prepared. OMVs with different pore sizes were obtained by liposome extruder.
3. To verify the surface display of Im7 protein, EcN engineered bacteria and OMVs exhibiting Im7 protein were incubated with purified CL7-sfGFP fluorescent protein, and the results were observed under a fluorescence microscope. However, the experimental results were not satisfactory.

July 2024

1. We made a review of the previous fluorescence verification membrane surface display protein experiment, and consulted relevant literature to improve our experimental scheme, including OMVs purification method, sample preparation method, etc., and carried out fluorescence verification experiment again (Fig.4).


Fig.4 EcN pCold-Inak-Im7 800 nm extrusion OMVs laser confocal characterization

2. We also learned another way to display targeted elements on the membrane surface through literature review: the z domain that can specifically bind to the Fc end of IgG antibody is displayed on the surface of EcN membrane. In this way, the obtained OMVs are incubated and bonded directly with the antibodies with Fc domain purchased by the company to realize the display of targeted elements on the membrane surface.
3. Construct the z domain expression plasmid (Fig.5).


Fig.5 z domain expression plasmid map

4. The CL7-mCherry red fluorescent protein expression plasmid was constructed to subsequently verify the efficiency of OMVs delivery to target cells in Hela cell lines.

August 2024

1. We transferred the ampicillin resistant Cas9 RNP expression plasmid and kanamycin resistant Im7 display plasmid into engineered EcN strains for expression, and investigated the transfection concentration ratio of the two to obtain the optimal protein expression level.
2. CL7-mCherry red fluorescent protein with surface showing Im7 protein was incubated with OMVs and then transferred into HeLa cells for culture, after which its delivery effect was observed under fluorescence microscopy (Fig.6).


Fig.6 Extruded OMVs with Im7 on their surfaces carry CL7-fused red fluorescent proteins into HeLa

September 2024

1. Conduct an OMVs gene editing efficiency validation experiment in Hela cell line (Fig.7).


Fig.7 Verification experiment of genome editing activity in T7E1 cells.

2. 2.Do the writing of Wiki upload content.

2. HP

April 2024

1. HuBu-4-CHN's first offline pitch in Room B200, Qinjin Teaching Building, Yangluo Campus, Hubei University







2. SynBio China 2024 China Annual Conference on Synthetic Biology and Biofufacturing in Qiantang District, Hangzhou City







3. HuBu-4-CHN Lecture within the School of Life Sciences, Hubei University in Room B200 and Room B211, Qinjin Teaching Building, Yangluo Campus, Hubei University







4. HuBu-4-CHN Hubei University Affiliated Kindergarten presentation in Classroom of Middle (5) Affiliated Kindergarten, Hubei University







5. HuBu-4-CHN Hubei University Affiliated Primary School preaching in Classroom of Class5, Grade2,Primary School affiliated to Hubei University









6. HuBu-4-CHN Wuhan 19th Middle Lecture in Class 1, Grade 1, No. 19 Middle School, Wuhan







7. 2024 Synthetic Biology Open Conference (Hubei Station) in Hubei University Library, Sirui Hall

May 2024

1. "Reading in Lake University, forging a new journey" reading speech contest preliminary in Lecture Hall 303, Manchester United College, Hubei University





2. National Laboratory Linkage Open Day activity in Room 402, Teaching Building 5, Hubei University & Laboratory, 4th Floor, College of Life Sciences, Hubei University







3. 2024iGEM South China Exchange Meeting in Shenzhen University Lihu Campus





4. "Reading in Lake University, Forging a New Journey" Reading speech contest finals in Lecture Hall No.3, Hubei University

July 2024

1. The 11th iGEMer Exchange Meeting in China in Xi'an Jiaotong-Liverpool University, Suzhou











2. 1st "International Symposium on the Application and Security of Escherichia coli online





3. Communicate with SYPHU-China about its team products online

August 2024

1. The third Synthetic Biology Innovation Competition in Anyungu International Convention Center, Guangming Day, Shenzhen







2. Handbook of Synthetic Biology Ethics, compiled online





3. "Together" Building the Future - 2024 iGEM Exchange Meeting in Central China in Huazhong Agricultural University





4. The first iGEM team dry experiment exchange meeting in Northeast Normal University online conference

September 2024

1. Hospital oncology research(Cooperation and exchange, security, education, start-up market -- comprehensive) in Department of Oncology, Wuhan First Hospital







2. "Synthetic Biology Lecture Hall"(Education + Safety) online





3. Sunny Home - Special People Preaching in Sunshine Homeland, Wuhan







4. Wuhan Ilerite Research in Wuhan Ilerite Biotechnology Co.,LTD

3. Art

March 2024

1. Design logo
2. Design and produce team uniforms (short sleeves, jackets)
3. Design team flag
4. Begin operating various platform accounts and continue to update (mainly WeChat public account)
5. Create extruder animation

April 2024

1. Assist HP in the Yangluo campus presentation
2. Design and produce team cultural and promotional materials
3. Collect and integrate project information from four teams, design and produce synthetic biology open conference event roll-up and team introduction roll-up
4. Accompany the team to the Synthetic Biology Open Conference and record

May 2024

1. Design team mascot Archie
2. Produce Archie promotional video
3. Meeting minutes for the reporting session
4. Translate post content

June 2024

1. Design the national joint laboratory open day team poster
2. Accompany the team to the event and record the process, fill in feedback forms
3. Follow HP to participate in the 2024 Wuhan Seed Industry Expo as a volunteer service

July 2024

1. Design CCIC team project poster
2. Create team PPT template
3. Accompany the team to CCIC and record the process
4. Design the poster for the first "International Symposium on the Application and Safety of ECN1917"
5. Design web page homepage image

August 2024

1. Design web page background image
2. Paint elements needed for the background image
3. Design exclusive Aqi image
4. Produce promotional video script, reshoot promotion video content, hand-drawn images needed for the promotional video.

September and October 2024

1. Beautify the wiki website
2. Shoot and make presentation video