Overview
“Be brave when necessary, and be decisive when there is no turning back.”
-- Comment on Fan Zhendong's victory in Paris Olympic Games
“Be brave when necessary, and be decisive when there is no turning back.”This comment is a good reflection of our mood in the process of project realization this year - actively changing the plan and thinking in difficult times, and constantly learning lessons from setbacks.
The characteristics of bioengineering mainly include modularity and standardization, design and construction based on engineering thinking, data-driven and computational modeling, and innovative ideas and reform schemes in practice.
Unlike traditional engineering disciplines such as construction engineering and vehicle engineering, bioengineering (synthetic biology) is full of instability and unpredictability in the process of practice, but it is also full of the possibility of breakthrough. ZJU-China2024 is excited to be iGEMers and make our contribution to the science of synthetic biology, which is full of possibilities. In this year's project, we hope to show the richness of fashion through our creative engineering, while showcasing our unique engineering design.
In the process of project implementation, we always adhere to the bioengineering iterative cycle of brainstorming, experimentation, and repeated discussion of possibilities, constantly learn from setbacks, and follow the "design-build-test-learn" cycle.
In the process of project implementation, we carried out various possibility prediction and experimental exploration, including RNA secondary structure prediction, phenotype observation, protein expression detection, luorescence expression and so on.
We construct protein-substrate binding models and microbial overall metabolism models through bioinformatics. (For more information, see the model page)
We have also developed user-friendly software that allows users to design the fashion totem they want. (For more information, see the software page)
We standardized the components involved in the project to meet the needs of the iGEM community. You are very welcome to use our section and contact us about how to use it. Any suggestions would be greatly appreciated. To learn more, click part.
Yield Improvement Module
Cycle1 - purified protein
Design
We just want to explore and enhance the catalytic activity of the enzyme vioE, so we want to purify a single vioE enzyme. We began to screen and experiment with different purification methods to select the most suitable purification method for vioE.
Build
In view of the wide application of his tag in protein purification experiments, we considered linking the vioE gene to the LacI inducible promoter commonly used in E.coli, and isolating the pure vioE enzyme by his tag on the advance marker after induction. At the same time, to verify the stability of our promoter, we attached sfGFP to the back of the vioE enzyme.
Test
Through the above method, we successfully induced the bacterial solution with green fluorescence using IPTG, and verified that the promoter could be correctly expressed. Subsequently, we obtained the correct expression of vioE enzyme by WB.
Study
The process of protein purification in the previous experiment was time-consuming and laborious. We hope to evaluate vioE's function by directly observing the phenotype.
Cycle2 - phenotypic observation
Design
A series of enzymes in the catalytic process of violacein were transferred into Escherichia coli to directly observe the phenotype - the amount of pigment production.
Build
We transferred a series of enzymes in the catalytic process of violacein into Escherichia coli, among which the enzyme vioE contains different mutants, and directly observed the phenotype - the amount of pigment production after induction. (See wiki protocol page for details.)
Test
When OD values were measured after induction, we did observe large changes in enzyme activity brought about by different vioE mutants. (See wiki result page for details.)
Study
The importance of different purification methods for different cell products is well understood. Looking ahead, we hope to develop methods for the extraction and measurement of most organic pigment synthases and their products.
Painting Module
Cycle1 - write in module based on split-cre-loxP
Design
We select the division site and design two different vector plasmids to accommodate our split-cre-loxP.(more detail of design please click)
Build
Using jump29 and jump42 as backbones, we constructed the Jump42-LoxP-TT-loxP-GFP plasmid storing loxP sites and the Jump29-LACuV5-creN-creC plasmid for split cre.
Test
After molecular cloning, and gene sequencing, finally we got the right plasmid after many failures.
Learn
However, such a design makes us find that the repetition of VVD at close distances causes great trouble in sequencing or molecular cloning, and poses serious challenges for subsequent functional verification
Cycle2 - Plasmid structure optimization
Design
We designed another two plasmids which two elements of the cre enzyme on two vectors with different replicons and resistance (pJUMP42-lac promoter-creC-promoter-loxP-TT-loxP-GFP and pET15b-lacUV5-creN) is induced by IPTG via Lac opreaon, and LacI expression was compensated by pET15 vector.
Build
We successfully constructed the relevant plasmids, and the sequencing results indicated this.
Test
We verified the feasibility by means of co-translocation of E.coli and colony PCR, which will also become the basic paradigm for our subsequent functional characterization, details can be seen on the result page.
Learn
We were able to observe phenotypes at the genetic level, however, there were still considerable intensity of leakage expression and unstable expression of splitting enzyme, which prompted further modification of plasmid structure
Cycle3 - Leakage expression risk exclusion
Design
We want to modify the distance from RBS to the start codon to make it easier to express the enzyme. In the meantime, the cap binging site in front of the GFP component promoter was deleted for less leakage expression.
On the other hand, considering that our modified enzyme works directly on DNA, we designed a PCR-based editing efficiency detection method to avoid the influence of fluorescence leakage expression.
Build
We successfully constructed the relevant plasmids with recombination, and the feasibility of the new method is simply tested.
Test
We cultured E.coli after co-transfer, and colony PCR verified the leakage level with low cutting activity, based on which we further performed light-induced characterization to measure its cutting efficiency. (more detail of design please click)
Learn
We successfully constructed a tool that can be photoinduced to edit genes, but only in response to one signal, and it inspires us to explore tool kits that can respond orthogonally to multiple signals.
Readout plasmid construction for mutated HYER characterization
Cycle 1-construction of readout plasmid
Design
To test if mutated HYER could block the expression of genes downstream, we put one loxp site with TRS/RS sequence on both sides between promoter and RBS, controlling the expression of sfGFP.
Build
Through homologous recombination, we inserted the TRS/RS-loxp-TRS/RS module into desired place.
Test
We checked the agar plate but found out that colonies were white.
Learn
After sequencing, we knew that there were a few bases missing(including ATG) upstream of sfGFP sequence.
Cycle 2 - rectification of readout plasmid
Design
We planned to insert the missing bases in two ways: do overlap PCR, or add those bases in a new primer and get the right DNA fragment with recognition site for another homologous recombination.
Build
As described above, we carried out overlap PCR and homologous recombination with fragment from regular PCR.
Test
Gene sequencing showed that the plasmid was right this time. Theoretically, the plasmid might appear green without any intervention. Actually, we picked green colonies on the agar plate and got the right sequence. But after first readout-HYER co-transformation test, we found out that in control group(just readout plasmid), only from those white colonies could we find right sequence.
Learn
We thought it was indicated that the plasmid wasn’t pure from the beginning(probably because there could be multiple plasmids in one bacterium though the copy number of our readout plasmid was small), so we had to dilute the bacteria solution and culture them on agar plate again to get single colony that seemed to be white.
Cycle 3 - purification and proof of fluorescence glowing of readout plasmid
Design
Since there're different copies of plasmids in our bacteria and the right plasmid tended to make colonies white instead of green, we decided to isolate those white colonies for plasmid purification.
Build
We repeated plate smearing with diluted bacteria solution, and picked relatively white colonies for purified readout plasmids and green colonies as control group of wrong plasmids.
Test
Via gene sequencing, we confirmed that the white colonies produced the right plasmids, and proved sfGFP was expressed by using fluorescence microplate reader.
Learn
Though the colonies still appeared to be low in fluorescence intensity, the bacteria solution indeed glowed fluorescent green under fluorescence microplate reader, so the readout plasmid was practicable for next step. We thought sfGFP might not work well as a direct fluorescence readout for our experiment because of low efficiency of the ribozyme and the fact that amount and characterization performance of sfGFP was not ideal, so we finally turn to qRT-PCR instead of fluorescence microplate reader as the final readou
Reference
[1] Light-Inducible Recombinases for Bacterial OptogeneticsMichael B. Sheets, Wilson W. Wong, and Mary J. DunlopACS Synthetic Biology 2020 9 (2), 227-235 DOI: 10.1021/acssynbio.9b00395
