Team:Fudan-CHINA/Improve
Improved parts: BBa_K2886004
This year, we present BBa_K2886004 as our improved part in the judging form, but actually we make improvement to another three parts.
BBa_K2886004 and BBa_K2886005 are both improved from BBa_E0020, which has 87 uses till 2017. And we designed them from BBa_E0020 with the technology BiFC (bimolecular fluorescence complementation).
BiFC is a new technology put forward in 2012 aiming to visualize the interaction of two proteins. If you connect these two parts with your two target proteins respectively, when two target proteins interact with each other, two parts of BiFC might well bind with each other and emit fluorescence for observation, which, in turn, proves the combination of your target proteins.
We split ECFP at the site A155 into two parts and get cECFP (coded by BBa_K2886004) and nECFP (coded by BBa_K2886005) which are the C- and N-terminal of ECPF. If these two parts of BiFC bind with each other, and they will emit fluorescence just as complete ECFP.
To demonstrate that our BiFC system works, we carry out a series of experiment.
Firstly, to prove it preliminarily, we implement experiment in E. coli BL21. To begin with, we transform E. coli with plasmids carrying BBa_K2886013 (leucine zipper + nECFP) and BBa_K2886012 (leucine zipper + cECFP) respectively. Because of the existence of leucine zipper, which can bind with each other due to electrostatic attraction automatically without out outer-membrane, transmembrane domain or ligand. Then, when they express enough cECFP and nECFP, we lyse the bacteria with ultrasound and mix them up to emit cECFP and nECFP to a same system. At last, we measure the fluorescence with plate reader compared with negative and positive controls. Figure 1 shows that our attempt does succeed and it might be reliable for more complicated conditions.
Moreover, to further demonstrate the reliability of our BiFC system, we also do some similar things in Hela cells. The difference is that we add outer-membrane and transmembrane domain to it as well as substitute the leucine zipper with GS linker. We transform the cells with these plasmids and then induce them with the ligand VEGF. When we observe the cells under fluorescence microscope, we also see the fluorescence, which is showed in Figure 2.
Besides, we collaborate with Jilin_China to make sure that our BiFC can work in different labs. To our credit, the outcome is more than fruitful. Click here to learn more about it.
Through these experiments, we fully demonstrate that our BiFC system works and prove the improvement of BBa_E0020 is a success. And BBa_K2886004 & BBa_K2886005 can used as labels for interaction of two target proteins and the location of two proteins in subcellular level, tremendously expanding the usage of the original part and equipping iGEMers with another powerful tool for biological visualization.
BiFC is a new technology put forward in 2012 aiming to visualize the interaction of two proteins. If you connect these two parts with your two target proteins respectively, when two target proteins interact with each other, two parts of BiFC might well bind with each other and emit fluorescence for observation, which, in turn, proves the combination of your target proteins.
We split ECFP at the site A155 into two parts and get cECFP (coded by BBa_K2886004) and nECFP (coded by BBa_K2886005) which are the C- and N-terminal of ECPF. If these two parts of BiFC bind with each other, and they will emit fluorescence just as complete ECFP.
To demonstrate that our BiFC system works, we carry out a series of experiment.
Firstly, to prove it preliminarily, we implement experiment in E. coli BL21. To begin with, we transform E. coli with plasmids carrying BBa_K2886013 (leucine zipper + nECFP) and BBa_K2886012 (leucine zipper + cECFP) respectively. Because of the existence of leucine zipper, which can bind with each other due to electrostatic attraction automatically without out outer-membrane, transmembrane domain or ligand. Then, when they express enough cECFP and nECFP, we lyse the bacteria with ultrasound and mix them up to emit cECFP and nECFP to a same system. At last, we measure the fluorescence with plate reader compared with negative and positive controls. Figure 1 shows that our attempt does succeed and it might be reliable for more complicated conditions.
Figure 1. The sketch map of BBa_K2886013 & BBa_K2886012 and the result of their interaction in E. coli induced under 4 ℃. (a) BBa_K2886013 & BBa_K2886012 can bind with each other through the electrostatic attraction of leucine zipper and emit fluorescence. (b) The background fluorescence of nECFP , cECFP and their average value (average(n,c)). Fluorescence emission of the mixture of nECFP and cECFP shows a 4.19 folds of intensity. And the original ECFP shows a 5.7 folds of intensity compared to original ECFP (BBa_E0020), exceeding the less than 10-fold result reported in literature.
Figure 2. The structure of BiFC in Hela cell and the results of tests. (a)We construct 4 plasmid, whose inner-membrane domains are A: 1 GS linker + cECFP, B: 1 GS linker + nECFP，C: 2 GS linkers + cECFP and D: 2 GS linkers + nECFP. (b)The fluorescence intensity of after adding VEGF (AB+) has a 1.43 folds compared with before (AB-) in the A+B group. And in C+D group, it’s 1.24 folds.(c)Relative fluorescence intensity at different cotransfection in A+B group. Relative fluorescence intensity is evaluated by grey level measurement of ImageJ. They all show apparent differences, so it’s safe to conclude that our BiFC system works.
Through these experiments, we fully demonstrate that our BiFC system works and prove the improvement of BBa_E0020 is a success. And BBa_K2886004 & BBa_K2886005 can used as labels for interaction of two target proteins and the location of two proteins in subcellular level, tremendously expanding the usage of the original part and equipping iGEMers with another powerful tool for biological visualization.
BBa_K2886007 encodes VEGF-165 with His9 tag. VEGF is a big family including lots of types. VEGF-121 was firstly introduced to iGEM by Slovenia in 2012 (BBa_K782061) and then was improved by SYSU-CHINA in 2017 (BBa_K2298001). VEGF-165 was also firstly introduced to iGEM by NYMU_Taiwan in 2012(BBa_K896922). However, there is no detail in the part wiki at all except for the part name, and actually we find this part using registry blast. This year, Fudan-CHINA makes new improvements to VEGF-165 to make it more useful and accessible to enrich the choices of iGEMers.
We perfect the description of VEGF-165 to make more iGEMers can know about and search for VEGF-165 expediently. And to test our STEP system, we need a large amount of VEGF-165. However, buying VEGF from companies will be really expensive. The average price of 1mg VEGF-165 is as high as 10,000 yuan (nearly 5000 dollars). Thus, we decide to construct plasmid and express VEGF-165 by ourselves. We design a sequence coding VEGF-165 followed by a 9×His tag and then entrust Sangon Biotech to synthesis it totally on the backbone pET28a. To our delighted, we succeeded in expressing VEGF-165 in E. coli and then purifying it. The result is showed in Figure 3.
Considering the successful results, we are willing to share this part with all iGEMers around the world to express and purify VEGF-165 on yourselves at an extremely low cost, enabling you to spare more funds for other experiments and human practice.
For detailed information，please click here to visit our Receptor Optimization page.
Figure 3. The gel of VEGF. The original VEGF is 38.2 kDa, and it becomes about 48 kDa after we adding the His-9 tag. On the gel, we can see a light band between 48.0 kDa and 63 kDa, which proves that we succeed in expressing and purifying VEGF-His9.
For detailed information，please click here to visit our Receptor Optimization page.
BBa_K2886010 is a repressible tet binding site improved from BBa_R0040, a commonly used part in iGEM. This time, we designed a mutation of tet binding site. According to the simulative result, BBa_K2886010 shows a higher bind affinity compared to the original TetR. Figure 4 exhibits the result.
For detailed information，please click here to visit our Receptor Optimization page.
Figure 4. The structure and ik_ball _wtd of wild type and mutant type tet binding site. (a) Structure of wild type (WT) tet binding site. (b) Structure of mutant type (MT) tet binding site. The transcription factor is colored to show its electrostatics distribution. Compared to WT, MT with a substitution of A to C on base pair 11 has a lower interface energy decreasing from-37.467 to -38.411 kcal/mol, tested by RosettaDock. (c) The most significant improvement is lk_ball_wtd (also known as orientation-dependent solvation energy), indicating MTmay have a higher affinity to the transcriptional factor.
BBa_K2886002 encodes VEGF-scFv which can recognize VEGF. VEGF-scFv was firstly introduced to iGEM by NCTU-Formosa in the year 2015 (BBa_K1694003). And this year, we make improvement to the VEGF-scFv to make it more suitable for engineering. In our STEP system, to improve the affinity of the VEGF-scFv to VEGF, we used Rosetta to help design our protein domain. However, we find that the initial version of VEGF-scFv in parts has no accepted structure in PDD (Protein Data Base). Thus, to improve the reliability of our results, we introduced a new kind of VEGF-scFv that has accepted structure in PDD to iGEM.
For more information, please click here to visit our best basic page.
Figure 5. The structure of VEGF-scFv and the interface score of modified version of BBa_K2886002 compared with original version and BBa_K1694003. (a) VEGF-scFv is two outermost Ig-like domains of the antibody of VEGF joined together with a GS linker. (b) According to the interface scores which indicate the affinity of VEGF-scFv to VEGF of revised BBa_K2886002, original BBa_K2886002 and BBa_K1694003, we can draw the conclusion that revised BBa_K2886002 probably has a higher affinity to VEGF after our modification.
[1]Wikipedia contributors. "Bimolecular fluorescence complementation." Wikipedia, The Free Encyclopedia. Wikipedia, The Free Encyclopedia, 12 Jul. 2018. Web. 17 Oct. 2018.
[2]Lai, Y. et al., Serum VEGF levels in the early diagnosis and severity assessment of non-small cell lung cancer. J CANCER 9 1538 (2018).
[3]Wikipedia contributors. "Vascular endothelial growth factor." Wikipedia, The Free Encyclopedia. Wikipedia, The Free Encyclopedia, 27 Aug. 2018. Web. 17 Oct. 2018.
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