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.
Figure 1. 4 represnets the sample in 4℃ and 20 represents the sample in 20℃. They are the mixture of nECFP and cECFP. Blank is the average of individual cECFP and nECFP.
As the results show, 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.
Figure 2. Fluorescence expression of positive control at different temperatures. The figure’s horizontal axis represents the temperature range (28℃ to 42℃), and the vertical axis represents fluorescence intensity of sfGFP. The red line is the sfGFP expression curve of NPC at 8th hours, the purple line is sfGFP expression curve of NPC at 6th hours, the blue line is sfGFP expression curve of MPC at 8th hours and the organe line is sfGFP expression curve of MPC at 6th hours.
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.
Here is the link to the wiki of Fudan-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.
Figure 3. The figure’s horizontal axis represents the time, and the vertical axis represents OD value. The blue line is the growth curve of Blank, the orange line is the growth curve of cob and the gray line is the growth curve of var1.
As the results shows, the two genes almost don’t have influence on bacterial growth. The result indicated that the two genes are not 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 ).
Figure 4. The bar colors blue, orange and gray represent the temperatures 31, 35 and 39°C, respectively. The height of the bars corresponds to the normalized fluorescence.
As shown in figure 4, 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 5).
Figure 5. Comparison of measurement results between Jilin_Chian and NPU-China. K2541003_J, K2541004_J, K2541010_J are the measurement results of Jilin_China, and K2541003_N, K2541004_N, K2541010_N are the measurement results of NPU-China. The height of the bars corresponds to the normalized fluorescence.
We are very grateful to NPU-China for helping us with this experiment.
Here is the link to the wiki of NPU-China.
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.
Here is the link to the wiki of NEU_China_A.
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 made an improvement that we did a site-directed mutagenesis of sfGFP (BBa_I746916) and got sfGFP（BbsI free）for Golden Gate assembly. We would like to constructed a standard part of sfGFP(BbsI free)(BBa_K2541401). 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(BbsI free). 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.
Figure 6. From left to right, each gel pore reprenents 1kb marker, plasmid + EcoRI + PstI, plasmid + PstI, plasmid + EcoRI and original plasmid.
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.
Here is the link to the wiki of TUST_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 7) works.
Figure 7. The plasmid backbone of device A.
PsiR is a repressor protein that binds to the pPsi promoter, 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:
Figure 8. Abs600 and fluorescence intensity once per hour in different psicose solution.
From the above results, we can see that with the increasing of psicose concentration, the fluorescence intensity increases, which matches 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.
Figure 9. The intermediate device for constructing standardized parts.
The plasmid was confirmed to be constructed as expectation by enzyme digestion (original plasmid, BsaI single digestion, and EcoRI and PstI double digestion). The agarose gel electrophoresis (figure 10) 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 short length ). 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.
Figure 10. Each gel pore reprenents 1kb marker, original plasmid, plasmid + BsaI and plasmid + EcoRI + PstI.
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.
Here is the link to the wiki of XJTU-China.
This year, we cooperated with Shanghai Tech. They helped us measure heat-inducible RNA-based thermosensors in cell-free protein synthesis (CFPS). CFPS is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. According to recent article reported, cell-free system is more sensitive to detect RNA behavior than traditional measurement method. With the help of CFPS, we can get more precise experimental results in vitro.
For cell-free measurement, we constructed a new measurement device with T7 promoter. Then we sent ten sets of plasmids to them. They expressed the ten samples in cell-free system under three temperatures: 29°, 37°and 42°. Then they measured the relative fluorescence intensity by microplate reader. Finally, we received their data and computed the fold-change of the response in the given temperatures.
Figure 11. Fold change of the heat-inducible RNA-based theromsensors. Each plot represents an individual theromsensor. The purple horizontal represents the fold change of positive control. The purple vertical represents the normalized fluorescence of negative control.
As these results show, the heat-inducible RNA-based thermosensors we designed can work in CFPS. The fluorescence value increases with temperature elevated. Besides, the difference in fluorescence intensity and the rate of increase points to the diversity in thermosensor response.
We appreciate ShanghaiTech to help us measure the heat-inducible RNA-based theromsensos. Hoping that we can maintain the long-time friendship and collaborate with each other in the future.
Here is the link to the wiki of ShanghaiTech.