Difference between revisions of "Team:OUC-China/Notebook"

After studying lots of papers and brainstorming for about one month, we confirmed our project for this year, which is about a regulation method at post-transcription level. We came up with the idea that controlling the quantitative expression of proteins by inserting different stem-loops into circuits.

Our wet lab, dry lab and also design group started to work!

We established our team consisting 12 students from different grades and different majors and then registered our team, OUC-China.

For our wet lab, we came up with problems when we organized the logical thoughts of our project therefore we went to Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences to consult professors.

We confirmed our pre-experiment design. Firstly, we needed to make sure that our parts like GFP and mCherry could work well. Then we did experiment to verify that the stem loop wouldn’t cause terminate, resulting in the downstream protein could express approximately constantly in circuit with and without stem loops. Moreover, we did pre-experiment to ensure that our dual fluorescence reporter system can work well.

Also, we started to learn how to operate some software, like Primer Premier, Snap Gene, Oligo, etc. to design our own circuits.

Figure1 The expression of our part GFP and mCherry

Figure2 The figure shows the fluorescent of downstream mCherry in the circuit with or without stem-loop, and there’s no significant difference between them(P=0.01). Error bars indicate s.d. of mean of all sequences.

After consulting professors and studying papers, we decided to use E.coli as our chassis.

We compared northern and RT PCR to decide with method would be more suitable for our project. We found that RT PCR was easier to operate, had more accurate data and finally we confirmed to use RT PCR to detect mRNA.
We also went to Peking University to communicate with the Peking iGEM team. We visited their own lab and both presented our project and exchanged our ideas. Moreover, we communicated about the team management and personnel recruitments and selection.

Figure3 In Peking University

We started to construct our circuits by ourselves. At first, we designed four circuits that both had the same promoter, RBS, terminator and two fluorescent proteins GFP and mCherry. In the first circuit, there were only proteins. In the second circuit, there was an endonuclease RNase cleavage site which would make the circuit into two fragments. Both in the third and fourth circuits, there was stem-loops but circuit 4 had an RNase site.

Figure4 The design of our first four circuits.

We divided our wet group into two parts to use enzyme cleavage and overlap respectively to construct our circuits.

We met problem that the BFP couldn’t transform or emit fluorescence. We thought that the part we used couldn’t work well so we decided to use GFP rather than BFP.
We publicized iGEM and synthetic biology in our school by organizing a lecture for about 100 people. We started to recruit new iGEM team member for 2017.
We also carried out our other parts of human practice, like holding summer camp for students from other universities.

We successfully used overlap to construct our circuit which included GFP and mCherry as two fluorescent reporter proteins.

Figure5 We constructed our circuit by overlapping

We also tried to insert stem loops in our circuit and we successfully annealed and extended the primer.

We learned the protocol of how to extract mRNA and started to do experiment from circuit 1 to circuit 4. Then, we detect the mRNA by using RT-PCR.
We also started to measure the growth curve of E.coli by using UV spectrophotometer, which under the wave length of 600 to decide the time point of detecting the expression of protein.

We got the results of the growth curve of E.coli and ensured that they would arrive logarithmic phase after 6-7 hours and arrive stationary phase after 21-22 hours.
We started to test the expression of GFP and mCherry in circuit 1 to 4 by using microplate reader.

Our wet lab group was divided into three groups: extracting mRNA, detecting the expression of protein and detecting the mRNA. We tested four circuits we had designed. We got the positive correlation between the quantity of mRNA and the free energy of stem loops.

Figure6 The correlation between the quantity of mRNA and the free energy of stem loops

At the same time, we designed more stem loops with various free energy and inserted them into new circuits.
For human practice, we went to Qingdao Science and Technology Museum to hold a lecture for about 100 people to popularize synthetic biology and the importance of quantification in our daily life.

Figure7 We held a lecture for the delegation from Tibet

For the dry lab, we wanted to get the relationship that the concentration of mRNA changed with the time. We firstly tried to understand the process of degradation of mRNA, the transcription rate of mRNA and the cleavage efficiency of RNase E.
For the wet lab, we continued to design new stem-loops and inserted them into circuits.

We had our data of the expression of proteins in four circuits which showed the relationship with the free energy of stem-loops.

Figure8 The protein fluorescence of upstream GFP to downstream mCherry of different circuits, background subtraction has been normalized with control group.Ratio = {[RFPterm/GFPterm]/[(RFPcontrol/GFPcontrol)mean]

Meanwhile, we held the summer camp for students from different universities. We introduced iGEM and synthetic biology to them by organizing lecture, operating experiment, designing their own posters.

We went to Guangzhou to attend CCiC and communicated with about 30 iGEM teams there.
We had two new circuits and three parts of our wet lab group started to test them. These two circuits had new stem loops with different free energy and we tried to further verify the relationship between quantitative expression of proteins and the free energy of stem loops.

Figure9 We had two designed stem loops which respectively had the same free energy with the origin one.

For our wet lab, we had another new two circuits and we tested the expression of mRNA and proteins as usual.
For collaboration, we collaborate with XMU 2016 to publish the survey of our project.

We focus on measuring the strength of our promoter, pBAD promoter. What’s more, we expanded our project to the three fluorescence reporting system and explore Gibson assembly to do this.
For collaboration, we continued contacting with HFUT-China, they helped measure our part sequence by their software and give us some suggestion about the genes and relevancy.

We started to standardized our parts and did collaboration with JNFLS 2016, they helped us to use flow cytometry to measure the fluorescence intensity of GFP and mCherry of circuit 3 according to our protocol.