Measurement of pBAD Promoter
NYU Shanghai made a Bacterial Music Generator this year, which can translate the colors and positions of bacteria colonies on a plate into sound composition. Integrating the unpredictable nature of life with digital technology, they aim to introduce new forms of bio-data into the sound domain. They engineered E. coli expressing chromoproteins, fluorescent proteins, or luciferase to produce colors visible under white light. We helped them test the strength of pBAD promoter, which they used to control and predicted the expression of target protein.
We characterized the inducible promoter (BBa_I0500) in August. We tried to test it in both liquid and agar medium, which can be more helpful to our friends from NYU Shanghai. And it is the first time the pBAD promoter was measured in LB agar medium in iGEM. We constructed a GFP expression device which consisted of the pBAD promoter (BBa_I0500) and a GFP coding sequence with RBS (BBa_I13504).
The liquid cultures were diluted to OD600 0.0015 in LB and added to the wells of a 96-well plate together with arabinose at 7 different concentrations (0%, 0.00002%, 0.0002%, 0.002%, 0.02%, 0.2%, 2%, m/V). The absorbance at 600nm and green fluorescence (excitation: 485 nm, emission: 520 nm) were measured every 30min in a plate reader, until the E. coli cells reached stationary phase (that is, the absorbance and fluorescence did no longer increase: about 8 hours from OD 0.5). Both absorbance and fluorescence were background (LB only) corrected. The fluorescence was then normalized for the number of cells by dividing by the absorbance.
The tested cultures were incubated at different concentrations of arabinose (0%, 0.002%, 0.02%, 0.2%, 20%, m/V). We put 150μL (OD=0.15) liquid culture into a 90mm-diameter plate contained 20mL LB agar medium. One hour later, a piece of 1cm-diameter sterile filter paper was put in the center of each plate. 15μL Ara liquid was put on the center of the paper. Eighteen hours later, the black and white photos were taken under 362nm excitation light, another ten hours later, the colorful photos were taken by a digital camera. It showed that 2%(m/V) Ara had the best result. Each concentration had three biological replicates. And you could have an object image of the fluorescence of GFP under different concentration of Ara.
Reference
Schaerli Y, Munteanu A, Gili M, et al. A unified design space of synthetic stripe-forming networks[J]. Nature communications, 2014, 5.
Assist in Survey of Chewing-Gum & GMOs
Aix-Marseille created an E. coli strain that produced enzymes, which could degrade rubber polymers. Nowadays, chewing gum is the second urban pollutant after cigarette butts. To clean chewing gum, specific machines are used. Be cause of heaviness and expensiveness, Aix-Marseille started their project.
They wanted to collect information about chewing-gum and GMOs worldwide. We were glad to help them to collect it. At first, we translated their questionnaire from English into Chinese carefully and checked it twice by two members of our team. Then, we went to Zhongshan Road and Taidong Business District away from our lab to interview the passersby one by one and randomly picked. The interviewees completed the questionnaire by themselves. At last, the results were submitted by us.
Because of our work, Aix- Marseille gave us a Gold Collaboration Badge, the highest level of honor. Here it is!
Assist in Modeling
This summer HFUT_CHINA designed a software named BioDesigner, which could help teams to construct their own genetic circuits and make the simulation online. It could also automatically provide teams with biobricks related to which had been used lately, and information about other teams’ experiences.
In order to give better simulation results and predictions to users, we helped them to improve their functions of the simulation part and deal with some detailed statistics. We also concluded the common use of ordinary differential equations (ODEs) and logistic models and assisted them in the parameter choice and analysis. Thus they enriched their simulation part with deterministic model and parameter analysis.
For the development of the software, we also gave our sincere advice about the User Interface (UI). It then advanced their optimization and made the software better.
Help from Peking
We introduced Gibson Assembly to our lab this year. During the processing, we obtained a lot help from Yihao Zhang, who is the team leader of Peking.
Help from Berlin
We also got a lot for the help from Berlin. Early in our project, we had some questions about character of ferritins and the details of mineralization. Berlin answered us meticulously and gave many useful advices. Finally, they sent us the parts including BBa_K143800, BBa_K143801, BBa_K1438025, BBa_K1438022, BBa_K1438027, BBa_K1438028, BBa_K1438031. However, we failed to transform the plasmid into E.coli, and we have to construct three parts by ourselves. We still thank our friends from Berlin so much, both their advice and cooperation spirits.
Raman Spectrometer of QIBEBT
The Qingdao Institute of Bioenergy and Bioprocess Technology (QIBEBT) was cofounded by Chinese Academy of Sciences (CAS), the provincial government of Shandong and the municipal government of Qingdao in 2006. QIBEBT is one of China’s primary national research institutions for renewable energy and green materials, focusing mainly on research and development of the resources, technologies, products and processes for bio-based energy and materials.
We used Raman Spectrometer there to test the protein expression of our devices under Yetian Su’s guidance. We do appreciate the favour of providing us with equipment and technical support.
Participation in iShare
We participant in iShare built by team Nankai, which is an all-round resource sharing platform. We shared 4 materials on iShare this year.
