Team:Tianjin/Engineering
Overview
The "Design-Build-Test-Learn" loop (DBTL) is a very important engineering framework. In the process of realizing the project, we also integrate this idea into our behavior, and constantly adjust the project, optimize the design and explore the future direction through feedback and iteration. Here is a record of our iterative process this year, showing the development of our project.
Pre-invastigation
At this stage, we mainly did brainstorming to choose the general direction and theme of our project.We consulted some articles on cell-free systems, and put forward the idea of constructing chromosome-free eukaryotic cell— CREATE.
Iteration 1
Design
① The delta site was selected as the cleavage target by consulting the literature.
② The original cutting plasmid was designed.
you can click link to the design page for details
③ The initial characterization method was designed: GFP gene was integrated into yeast chromosome.④ A mathematical model was designed to describe the change of the number of cells with respect to time.
Build
① Construction of the first cleavage plasmid Δ g.
② GFP was integrated into chromosome.
③ Based on Logistic model, some known yeast-related constants (generation time, etc.) were used to predict the number of cells in advance.
Test
① 4742 yeast was cleaved with the initial cleavage plasmid Δ g.
② Comparing the parameters predicted by the model with those calculated by the experimental data, there is a big deviation in the data when it is close to the environmental capacity.
Learn
① We found that cas9 using pGal promoter may have leak expression problem, which makes it difficult for us to obtain successfully transformed yeast colonies from the culture medium after transforming cutting elements into yeast cells. (The transformed yeast is difficult to grow because the plasmid will be partially cut even if it is not induced after entering the cell.)
② Logistic model has better fitting effect on environmental capacity and in logarithmic growth period, but it needs to be further optimized for specific growth environment.
Iteration 2
Design
① In order to strictly control the expression of cas9 and further improve the chromosome cleavage efficiency, we designed version 2.0 of the cleavage element: 7flip + cre cleavage system .
you can click link to the design page for details
② Other methods to characterize the formation of CREATE were designed.you can click link to the design page for details
③ After consulting the literature, we consider that the growth rate of cells can be described by the change of organic matter content in cell culture medium.④ Refer to the previous IGEM team's modeling and related papers, and predict the degradation reaction by differential equation.
Build
① Construction of a new cutting element .
② Refer to Monod model for continuous culture.
③ It is assumed that the degradation of proteins accords with the first-order kinetics.
Test
① Characterization of new cutting elements.
② Two different cutting elements are compared. For example, the growth ability of yeast after transferring different cutting elements was tested. We also made further comparisons through flow cytometry .
Learn
① Through experimental verification, we can show that the newly constructed cutting element is more effective than the original cutting element.
you can click link to the result page for details
② By designing a double induction system, we have carried out more strict regulations on cas9.③ When characterizing the new cutting element, we found that the half-life of the original GFP was too long to distinguish the CREATE from the normal cells in a short time
④ Monod model is good for describing the exponential growth period of yeast cells, but it can only be used to predict the number of cells, but can not predict other useful information;
⑤ Monod model, which predicts cell growth through nutrient change of cell culture medium, provides a new possible method for measuring cell number.
The predicted half-life is only relatively close, not accurate.
Iteration 3
Design
① By consulting the literature, we know that Saccharomyces cerevisiae SY14 has only one chromosome, and we imagine that this yeast with only one chromosome may form chromosome-free cells more easily, which will improve the formation rate of CREATE.
② A GFP with a shorter half-life is designed.
③ Design an experiment using fluorescence microscope.
you can click link to the design page for details
④ An experiment was designed to verify whether the enzymes of chromosomeless cells are still active.you can click link to the result page for details
⑤ It is known from literature that the estimation of chromosome-free cell formation rate by the method of discrete but discontinuous equation is closer to the real situation.⑥ The reaction count of protein degradation reaction needs to be measured experimentally. By trying to fit the curve, the reaction count can be fitted.
Build
① We found the yeast strain SY14 and transferred it to the new version of the cutting element.
② We constructed fGFP that degrades rapidly and integrated this segment into the chromosome.
③ Transformation of mcherry into the engineering bacteria.
④ Based on Lotka-Volterra population competition model, an evolution rule description set by cellular automata is constructed.
⑤ The curve of fluorescence intensity changing with time was fitted, and the reaction count was calculated by physical and chemical related formulas.
Test
① We used flow cytometry to compare the rate of chromosome-free cell formation between SY14 and conventional strain 4742.
② Test whether the half-life of fGFP is shortened.
you can click link to the improvement part page for details
③ Use a fluorescence microscope to observe the disappearance of fluorescence signals. ④ We compared the chromosome-free ratio predicted by fitting curve with the experimental data of flow cytometry sorting. ⑤ We model the initial fluorescence intensity, extrapolate it reasonably and compare it with the follow-up data, and the prediction effect is good.Learn
① We were surprised to find that SY14 with only one chromosome was less efficient than 4742 with 16 chromosomes.
you can click link to the result page for details
② We visually observed the disappearance process of nucleic acid dye and fGFP using fluorescence microscope.you can click link to the result page for details
③ Cellular automata model is very helpful in describing cell growth, especially in co-culture system.④ The degradation time of fGFP was greatly shortened; Although protein degradation reaction is not a simple first-order reaction, it can be used to estimate the half-life through the first-order reaction.