Difference between revisions of "Team:Jilin China/Collaborations"

1.Fudan_China

We have established a good relationship with Fudan_China this year.

This year the project of Fudan_China is the STEP receptor engineered according to the predecessors. The receptor consists of two chains, one chain is TC chain with a transcription factor and another chain is PC chain with a protease. To test whether transformation and construction of these two chains was successful, they replaced the transcription factor and protease downstream of the two chains with a pair of complementary semi-fluorescent proteins that they self-designed disassembled ECFP.

In order to test whether their ECFP splitting successfully and whether it can apply to other laboratories, our team helped Fudan_China characterize these parts and verified that their design and transformation of ECFP on bimolecular fluorescence complementation is successful in E.coli. By doing so, they can know whether their disassembled ECFP works as expected, and whether the transformation and construction of PC chain and TC chain is successful. The result is very important for their project.

We inoculated 100ul bacteria solution containing pGEX-4T-1-ECFP, pGEX-4T-1-LZ-cECFP, pGEX-4T-1-nECFP plasmids and 50ul bacteria solution containing pGEX-4T-1- LZ-cECFP, pGEX-4T-1-nECFP plasmids to mix. After incubating for 3-4 hours, we made the initial OD = 0.1. When OD was close to 0.6, we added IPTG at 1:1000 to induce protein expression at 16-20°C overnight. We lysed and centrifuged the four groups of bacteria to get supernatant protein mixture. Then we placed the supernatant of cECFP and nECFP mixture at 4 ℃ and 20℃ to induce ECFP fussion. We measured the fluoresence of these 4 types of ECFP.

As the results shown, the fluorescence density of nECFP and cECFP mixture is higher than blank, which has significant difference between two groups according to statistic analysis (P<0.05).

They also helped us to do some verification work. We designed and constructed hundreds of RNA-based thermosensors this year. We need to measure and get their melting temperature. Temperature dependence of global factors such as the fluorescence parameters of GFP or the activity of RNA polymerase may contribute to the individual thermosensor measurement. They should, however, affect all thermosensors in a similar fashion. Therefore, we focus on the relative difference in the thermosensor activities and we need a positive control to normalize the effects of different temperatures. So we designed two positive control (called NPC, MPC) with no thermosensor sequence to make it less sensitive to temperature. In order to ensure the accuracy of our experimental results, Fudan_CHINA helped us characterize and verify the sfGFP expression of our positive control at different temperatures.

It can be seen from the results that the sfGFP expression of our positive control changes approximately linearly with increasing temperature. This is consistent with our own measurement. The results are vital to us because we need to use this positive control when we process the measurement data of each thermosensor. We are very grateful to Fudan_China for the experiment, which is helpful for us to carry out the follow-up experiments and the data processing.

2.NPU-China

We cooperated with NPU-China this year, a team from Northwestern Polytechnical University in China.

Our team helped NPU-China characterized two parts because their team was short of staff to characterize all parts. The two parts are coding sequences of Saccharomyces cerevisiae mitochondrial genome. The cob codes cytochrome oxidase b which is the composition of the electron transport chain. And the introns of cob were all deleted. The var1 codes ribosomal protein. We helped them measure the growth curve to verify if the two parts are toxic to cells.

NPU-China also helped us a lot. We designed hundreds of RNA-based thermosensors this year. We need to get their melting temperature, intensity and sensitivity. We have measured some heat-inducible RNA-based thermosensors by ourselves. Nevertheless, we know that at different laboratories and under different experimental conditions, the experimental results could differ. To ensure the accuracy of our experimental results, we sent 5 samples to NPU-China. They helped us measure sfGFP expression at different temperatures. (The 5 samples: RT1-4, RT1-33, K2541003, K2541004, K2541010 ).

As shown in figure W, the fluorescence intensity of K2541003, K2541004 and K2541010 increase with elevated temperatures. While the fluorescence intensity of RT1-4 and RT1-33 don’t increase with elevated temperatures. This is consistent with our own measurement results (figure J).

We are very grateful to NPU-China for helping us with this experiment.

3.NEU_China_A

This year our team and NEU_China_A sent experimental materials in the Distribution Kit each other. These materials greatly helped both sides. We sent Nitrate reporter: PyeaR-GFP composite (BBa_K381001) and Test Device 4 for interlab (BBa_J364007) to them. PyeaR is a nitrate and nitrite sensitive promoter and NEU_China_A replaced GFP with a blue protein. The promoter PyeaR expressed the blue protein under the induction of nitric oxide. So they can use this part (BBa_K381001) to detect nitric oxide. NEU_China_A sent superfolder GFP (BBa_I746916) to us for part improvement. This year, We used sfGFP_optimism (BBa_K2541400) as an improvement part. Compared with iGEM superfolder GFP (BBa_I746916), it has no BbsI restriction site and can be used for Golden Gate assembly. So we used the part (BBa_I746916) from NEU_China_A to verify that it contains BbsI restriction site.

4.TUST_China

This year, we built a friendly relationship with TUST_China. During the meet-up in NKU and CCiC, we exchangd ideas about projects.

TUST_China focused on tetracycline detecting and degradation this year, so they would like to collect polluted water in most places in China. There is a river called Yongchun River which is closed to several bio-product factories near Jilin university. We provided a water sample from Yongchun River to TUST_China so they can detect antibiotic pollution to enrich their projects.

Back again to our SynRT, we used sfGFP_optimism as our reporter protein in the measurement device. Due to the high quality of sfGFP_optimism, we would standardize it and compete for best basic part. However, after several trials, we failed in ligation, then we decided to request TUST_China to help us. We sent them our plasmid for fluorescence measurement and primers for the construction of sfGFP_optimism. Luckily, they finished the construction for us and provided useful suggestions in ligation: you can increase the ratio of insert to vector, from 3:1 to 10:1. After the construction, they sequenced and sent it to us, which was correct.

We really appreciated it that they helped us in such an important experiment. Hoping that we can build long-time friendship and work for synthetic biology.

5.XJTU-CHINA

This year we collaborated with XJTU-CHINA from Xi'an Jiaotong University and we helped them measure fluorescence, so they could get the relation between the concentration of psicose and fluorescence intensity. Furthermore, the relation between different concentration of psicose and pPsi promoter activation efficiency was obtained. We helped them verify that their Device A (figure O) works.

PsiR is a repressor protein that binds to the pPsi promotor, rendering it inactive. Psicose and PsiR repressor proteins are combined to form a negative regulation induction system (similar to the IPTG and LacI), and psicose is added to the system to induce EGFP expression.

When they measured the Device A, the results were not quite as expected. They suspected that both LB culture medium and psicose in the system may probably make the bacteria grow. What’s more, the amount of psicose solution added to the system is not consistent, so different osmotic pressure may lead different growth conditions of bacteria. When our team did the measurement, we cultured the bacteria to OD=0.6 and then re-suspended the bacterial precipitation with MM culture medium. We cultured the bacteria with different concentration of psicose and measured the Abs600 and fluorescence intensity once per hour.

The results are as follows:

From the above results, we can see that with the increasing of psicose concentration, the fluorescence intensity increases, which basically accord with their assumption.

XJTU-CHINA has also helped us a lot. For our experimental design this year, we have lots of RNA-based thermosensors to submit to the part registry, which is a huge and challenging task. If we use the traditional construction method, the time and cost of constructing these parts will be a huge problem for us. Fortunately, we met XJTU-CHINA at the CCiC conference, and they would like to help us design an intermediate device for constructing standardized parts.

In combination with their own experience in using Golden Gate assembly, they inserted an only 60bp DNA fragment into pSB1C3 plasmid. The DNA fragment contains BsaI restriction site. Golden Gate assembly will enable fast and efficient parts construction. They sent the device to us as quickly as possible so we could complete the construction of about 50 parts before deadline, which greatly reduced our workload, improved our experimental efficiency, and solved our problem.

The plasmid was confirmed to be constructed as expected by enzyme digestion (original plasmid, BsaI single digestion, and EcoRI and PstI double digestion). The agarose gel electrophoresis (figure H) showed that these enzyme-digested products were all normal, and the brightness was also normal (5bp and 60bp fragment cannot be fully displayed on the electropherogram due to its length is very short). Furthermore, they sequenced it. The sequencing results showed that the ligation of plasmid and insert was correct, and the sequencing results were of good quality.

In our follow-up experiments, the constructing device was fully applicable, and the success rate exceeded our expectation, which greatly reduced our workload. We constructed all basic parts before deadline. After this cooperation, we have established a deep friendship with XJTU-CHINA. We often discuss some problems encountered in Golden Gate assembly. We also discuss some questions about modeling.