Team:ZJU-China/Design/Toxinmanufacture

• Bacteria solution PCR: OrfX. One positive clone of 20 samples.

• DNA sequencing: OrfX.

Dynamic Light Scattering(DLS) experiment to measure the particle size of embedded E.coli.

Took photos and prepared for the banner~

The sequence of metK and tcdA1 were confirmed, which marked the success in standardization of 2 parts!!

Bacteria solution PCR: metK and OrfX. Both had positive clones. DNA sequencing: metK and OrfX. T4 ligation and transformation: plu0840+pSB1C3, plu1537+pSB1C3, tcdA1+pSB1C3. Selected from single colonies: CDS-tcdA1.

• Selected from single colonies of metK and OrfX.

• Submit the safety form. Selected from single colonies: CDS-plu0840. Double enzyme digestion and DNA gel extraction: plu1537, tcdA1, plu0840. Plasmid extration and double enzyme digestion: RFP. Protein extraction and SDS-PAGE: tcdA1, plu1537, plu0840. T4 ligation and transformation: plu0840+pSB1C3, plu1537+pSB1C3, tcdA1+pSB1C3. Bacteria solution PCR: CDS-plu0840.

Seamless assembly: plasmid+CDS-tcdA1, plasmid+CDS-plu0840. DNA gel extraction: tcdA1. PCR: device-plu0840.

The sequence of ermE+plasmid, Frr+plasmid and plu1537+plasmid were confirmed, which marked the success in standardization of 3 parts!!

• Selected from single colonies of metK and OrfX.

• Transformed standard plu1537 plasmid into BL-21. Bacterium solution PCR: ermE and Frr.

• We kept a Madagascar cockroach as our pet~

• Selected from single colonies of ermE and Frr. Extraction of plasmid (standard plu1537).

• Bacterium solution PCR: standard plu1537.

• DNA sequencing. Seamless assembly: plasmid+1537, plasmid+tcdA1-L+ tcdA1-M+ tcdA1-R, plasmid+ermE, plasmid+Frr.

• Successfully booked the HI-Boston Hostel and paid the full payment~ Seamless assembly: plasmid+metK, plasmid+OrfX.

• Extraction of plasmid mCherry and double enzyme digestion. Seamless assembly: plasmid+1537, plasmid+tcdB1. PCR: ermE, Frr, metK and OrfX. Clean up and DNA gel extraction.

• Capsulated Streptomyces Avermitilis toxicity test. TT01 toxicity verification.

• PCR: ermE, Frr, metK and OrfX. Clean up and DNA gel extraction.

• Again used flow cytometry to check the diameter of the embedded E.coli and tried to count the E.coli which were successfully embedded. The results were not that ideal again.

• PCR: ermE, Frr, metK and OrfX. Clean up and DNA gel extraction.

• Finished the background of avermectin and toxic protein. Sent bacterium sample to BNU and we would continue to help them.

• PCR: CDS tcdB1, CDS 1537, tcdA1-L, mcherry. DNA sequencing.

• Half-done the model of degration of toxic protein. Got the kit for detection avermectin.

• PCR: CDS tcdB1, CDS plu1537 and mCherry. DNA gel extraction: tcdA1-L and plu0840L. Seamless assembly: plasmid+pBad+plu1537.

• Detected the relationship between distance of food source and food consumption.

• Designed the primers for Frr, metK, OrfX and ermE again because the need for standardization.

• Preparation of competent cells.DNA gel extraction.

• Verification of the bait attractiveness.

• PCR and clean up. Concluded the successful PCR parameters of different gene clusters.

• Test the Avermectin detection kit with pure Ivermectin.

• Designed the primers for plu0840, plu1537, tcdA1 and tcdB1 again bucause of the need for standardization.

• Overlap: 0840L and 0840R.

• Tried to purify the embedded E.coli with the E.coli which were able to express RFP.

• Used flow cytometry to check the diameter of the embedded E.coli and tried to count the E.coli which were successfully embedded. The results were not that ideal.

• PCR: pBad, tcdA1-L, tcdA1-M, tcdA1-R, tcdB1, 0840L, 0840R, 1537, GFP.

• PCR: ligated product. Optimized the PCR program. Cleaned up. PCR again.

• SEM scanned our samples to verify whether Streptomyces avermitilis were successfully embedded with CNC.

• Fed the termites with Photorhabdus luminescens TT01 bacterium cells.

• PCR: pBad, tcdA1-L and GFP.

• Used Bgl2 and EcoR1 digested inserted fragment and cleaned up the product. Ligating fragment into plasmid with T4 ligase.

• Specified the standardization requirements and laid the foundation for primer designing.

• Mixed blue-stained termites with red-stained termites to verify trophallaxis.

Visited Shanghai Science&Technology Museum and promoted our Synbio-cards(Polypoly card).

One step cloning protocol finished. Designed the questionaire about termites.

PCR: pBad, GFP, tcdA1-M, tcdA1-R.

• Drawing plasmid circuits in snapgene.(toxic protein)

• Stained 50 termites in blue and 50 in red for trophallaxis experiments.

Completed the One Step Cloning protocol.

PCR: pBad, tcdA1-L, tcdA1-M, tcdA1-R, tcdB1, plu0840L, plu0840R, plu1537 and GFP.

• Listed 15 main problems we were confronted with, including safety, communication between dry group and wet groups.

• Designed a new method to embed the bacterium with CNC.

Designed the primers for plu0840, plu1537, tcdA1 and tcdB1 again bucause of the bug of extra RBS.

Discussed helping BNU and HUST. Designed and alpha tested the Synbio-cards. Improved the modeling.

Attended NCTU meetup in Taiwan. Present our half-done project and communicated with other teams. Decided to help BNU and HUST.

• Listed and detailed the wiki requirements.

• Fed the termites with Streptomyces Avermitilis bacterium cells.

Prepared the home page and member page of our wiki. Prepared the poster for NCTU meetup.

Prepared for the souvenirs for NCTU meetup.

Mark-recapture method failed because we failed to recapture enough stained termites.

Exams of summer semester.

• Completed 2 safety questionaires.

• Prepared to learn confocol to verify the CNC package for E.coli.

Put 150 blue stained termites back into the nest for mark-recapture method.

TEM scanned our samples.

Fed the termites with Photorhabdus luminescens TT01 bacterium solution to test the natural toxicity. Stained 150 termites in blue for future experiment.

TEM and SEM scanned our samples.

Fed the termites with Streptomyces Avermitilis bacterium solution to test the natural toxicity.

Extracted the genome of Streptomyces avermitilis.

Went to shanghai for the interview of visa. 12 members successfully passed the interview.

• Paid for the air tickets.

• Got the primers of toxic protein.

Optimization of the model of termites finding food.

Frozen the spores of Streptomyces avermitilis.

• Discussed the characterization of E.coli embedded in CNC, including TEM, SEM, confocal, FTIR and flow cytometry.

• Our proposal had been accepted and our team would be awarded financial support from SYNENERGENE! Cheers~

• Brainstormed how to do human practice and use the €5000. We came up with the Synbio-cards and Synbio-workshops.

Began to book the air tickets. Booked the first primers of toxic protein for one step cloning. Found the sequence of the gene we need in the Streptomyces avermitilis.

SEM scanned our samples again. Debugged the first modeling.

• Repeated the cellulase activity detection experiment. Meanwhile we measured the lysozyme activity in different parts of termites guts.

• 22nd Group meetig. Specified the medal requirement. Began to prepare for the NCTU meetup.

• SEM scanned our samples.

Completed a model of a cartoon termite holding a sword. Cleaned up our lab and rearranged all the experiment materials we would need.

Got the solid CNC with lyophilization. Built the framework of the first web page.

Learned the skills of culturing Streptomyces avermitilis in the professor's lab.

iGEM Shanghai Tour. Took part in the conference held in NYU. Visited FDU and communicated with them.

• Finished the protocol about TT01.

• Measured the cellulase activity in termites head, forgut, midgut and hindgut.

Detailed the medal requirements depending on our project. Finished the protocol about Streptomyces avermitilis.

Got the E.coli strain ET12567 from Professor Li's Lab. Began the experiment about Streptomyces avermitilis.

Read paper and discussed how to enhance the efficiency of Avermectin in Streptomyces avermitilis.

• TEM scanned our samples: using CNC to pack the Bacillus subtilis.

• Completed the cover and comic of our proposal which would be handed in to SYNENERGENE.

• Contacted with Hangzhou Termites control Institute.

• Got the genome of tcdA1 and tcdB1 in NCBI.

• Got TT01(a species of Photorhabdus luminescens) which could produce toxic protein. It would play an important role in our plan B.

Successfully checked in termites and tc protein family.

Successfully got the solution of CNC!! And it appeared Tyndall effect.

Exams of spring semester.

Our termite nest was sent out from Guangdong Province.

• Discussed the plan for embedding baterium with CNC and the self-assembly of CMC and chitosan on the surface of baterium.

• Debugged the problem of synthesizing avermectin in Streptomyces avermitilis. Consulted professors specialized in synthesizing avermectin in Streptomyces avermitilis.

Consulted professors specialized in termites and Bacillus subtilis.

Discussd the advantage of Bacillus subtilis campared with E.coli and the utilization of spore of Bacillus subtilis.

Consulted professors specialized in termites.

Consulted professors about directly embedding baterium with CNC rather than with the aid of protein domain. Our idea was confirmed and praised.

Communication with our instructor. Searched for information about cellulose binding domain(CBDs) and overexpression of ivermectin and avermectin.

Preliminarily designed the circuit in Streptomyces avermitilis.

7th Group meeting. Began to kill some undoable ideas and further dug the existing ideas, such as biological enigma machine and killing termites.

Team application completed.

Communicate with Zhejiang Science&Technology Museum.

Visited the AST space of Zhejiang Science&Technology Museum. Decided to collaborate with.

Concluded the winter project.

Everyone read wikis, learned something from previous projects and attempted to come up with new ideas.

1st meetup. Had a nice meal~