Team:Fudan-CHINA/Demonstrate
Demonstration
"No road of flowers lead to glory."
From our dedicated work in both wet lab and dry lab we’ve successfully proved that our STEP system can work in human cells and can be tuned to achieve an ideal output.
Figure 1. Basic theory of STEP.
At the beginning, we improved a previous part of ECFP (BBa_E0020) to be BiFC components (BBa_K2886004 & BBa_K2886005), verified their reliability and employed them to test cell membrane localisation and binding ability of STEP (Figure 2). With cyan fluorescence observed in both circumstances, we can confirm that both our developed parts and the first part of STEP system works appropriately.
Figure 2. BiFC tests. The BiFC system we designed was examined and used to test the localisation and extracellular domain of STEP. (a) Leucine zipper was attached to N- and C-terminal of split ECFP to test the reliability of BiFC. (b) Both terminals were attached to the transmembrane linker of STEP to see if ligand-inducible dimerization could take place. (c) Results of BiFC. Fusion proteins are expressed in E. coli BL21 separately. They were then released from the cells and incubated for maturation. The relative fluorescence intensity of split ECFP measured via plate reader is higher than the 10% of whole ECFP we expected, which is the normal result of BiFC. (d) VEGF-STEP localisation and dimerization test. Plasmids containing both chains of cECFP and nECFP are transfected into HeLa. A few significantly activated cells were observed under microscope, and the total fluorescence intensity of VEGF(+) group is higher than VEGF(-) group, indicating that our receptors are able to correctly locate and bind at the presence of VEGF. (*: P<0.05, ***: P<0.001)
For another part of STEP, we proved the effectiveness of our transcription factor tTA and promotor pTight (Figure 3), and constructed a stable cell line with pTight-mCherry in both HEK293T and HeLa.
Figure 3. Transcription Factor Test. (a) Fluorescent images of transfected cell with pTight-mCherry (-) and tTA-EGFP and pTight-mCherry (+). Photos were taken 48 h after transfection. (b) Relative fluorescence intensity in conditions above.
After both two sections of STEP were verified, we carried out experiment to confirm the whole system works and to explore the best condition for it to operate. We successfully figured out a relatively ideal transfection condition of TC/PC = 12 and total quantity of 2.44 μg per ml (Figure 4).
The results from our overall tests are confirmed by our model, which also suggests that there could be a certain set of TC/PC ratio and quantity that can lead to a best dynamic range which may be significantly higher than other conditions. Thus we believe that STEP can be further optimised through more subdivided gradients.
Apart from VEGF, we carried out experiments with D-Dimer-STEP, and achieved a larger dynamic range (Figure 5a). The VEGF-STEP was also tested in HeLa and extremely low background noise was observed during experiments (Figure 5b).
Figure 4. Transcription ratio and quantity test in HEK293T. (a) Relative fluorescence intensity at different PC concentration. The dynamic range increases as TC/PC ratio rises, while the gene expression drops after 14. (b) The 1.00 indicates the original quantity of 3 μg/ml TC and 0.25 μg/ml PC. . The best dynamic range is found in 0.75-fold, presenting a 4.90-fold of reporter expression level.
The results from our overall tests are confirmed by our model, which also suggests that there could be a certain set of TC/PC ratio and quantity that can lead to a best dynamic range which may be significantly higher than other conditions. Thus we believe that STEP can be further optimised through more subdivided gradients.
Apart from VEGF, we carried out experiments with D-Dimer-STEP, and achieved a larger dynamic range (Figure 5a). The VEGF-STEP was also tested in HeLa and extremely low background noise was observed during experiments (Figure 5b).
Figure 5. STEP system test for D-Dimer and in HeLa. (a) Test of D-Dimer-STEP in HEK293T. 4 paralleled experiments of TC/PC = 12, total quantity = 3.25 μg/ml test were done using the D-Dimer-STEP, and the dynamic range reached 3.17 folds. (b) Test of VEGF-STEP in HeLa. 4 paralleled experiments were carried out, but the dynamic range was only 1.49 folds in HeLa cells.
In our dry lab, to improve the binding affinity, we have redesigned 3 receptor binding domains: VEGF-scFv, KDR Ig domain 3 and D-Dimer-scFv (Figure 6). Also, we have redesigned the DNA sequence of the tTA promoter, to enhance downstream expression (Figure 7).
Figure 6. Binding energy variation after redesigning.
Figure 7. An example for designing result (tTA promoter).
Based on theoretical calculation of Gibbs free energy, we can now design or redesign receptors in silicon to get maximum binding affinity with the ligand. With input structure of receptors and ligands, we can find out the interface of the ligand-receptor complex, calculate binding energy, and mutate some residues to improve it. Even if there is only sequence information, homology modeling can be used to predict the structure. We believe, with the input sequence or structure of any ligand, we can redesign, or do de novo design of the sequence of receptor, thus extending additional functions of our system.
Figure 7. Computational structure analysis of protein complex
For detailed results from dry lab, please visit our results page.
Address
G604, School of Life Sciences, Fudan University
2005 Songhu Road, Yangpu, Shanghai, China
2005 Songhu Road, Yangpu, Shanghai, China
