Team:Hong Kong HKU/Description

HKU EVOLVE - Description

Medical Safe Opioid synthesis (Dr. Nikolai Eroshenko’s suggestion). Gene Lysis Circuit with Hin Recombinase. Industrial Biofermentation and biotransformation reactions Biofilm based bio-production (e.g. ethanol/ 2018 WHU) HKUST collaboration: for incompatible peptide synthesis Environmental Nitrate and ammonium oxidation pathway separation. Plastic degradation PETase and MHETase separation. CityU collaboration for 4 different plastic degradation pathways Oil Remediation Description Building our system with 3 simple steps 1. Standard Plasmid Construction 2. PCR Gene Loci Flanking Region. Cell with Lambda Red Plasmid. Selection of positive Clone. Now cells will have single copy of the gene inside the genome. The copy dictates the cell’s phenotype. 3. Induction of Recombinase When lox sites faces in opposite directions, the DNA inverts. A equilibrium position would be achieved. Or in high Cre, it has small deletion chance for opposite circuits. When lox sites faces in parallel directions, the DNA is deleted. In a pilot experiment, a convergence to 50:50 ratio is hypothesized even with different metabolic burden of 2 systems. See dry lab for simulations. QS based consortium regulation might be hard to broadly apply to metabolic engineers[2]. Therefore, we aim to design a system that is easy to use, broadly applicable with most genetic circuits with high stability facing different variations. Besides the mutant Cre lox system, there are also recombinase systems that may work in our hypothesis. 3 Simple Rules on how to design the circuit P = promoter. | = double terminators. Brackets = lox sites. Letters = gene Invert = inverted gene. 3 rules 1. All lox sites are orthogonal 2. No overlapping of lox sites 3. Every bracket inside another bracket increase in 1 level {(A|| ) } with level 1{} and level 2 (). For every higher level bracket, it generally have 1/2n probability of expression. So A:B:C would be 1:1:2 in ratio. Metabolic differences and Intermediate Zone What if one phenotype grows much faster? By introducing equilibrium position, if, for example orange (phenotype B) grows faster, when there are more orange, it is also more probably for B to transit to A due to a larger number. Hence, a equilibrium position is introduced, making the ratio not very sensitive to metabolic differences. Check out the dry lab simulations for more details! What if it is in the transition state? The transition state does make the system sometimes non-functional, if the intermediate zone of both transcriptome may make the cell have decreased growth rate and, in some case cell death. We aim to solve this problem via using a crRNA array that targets the transcriptome of the other phenotype. Pairing with decradation tag, decreases the overlapping time between phenotypes! What if the lox sequenced is excised? To deal with this problem, for long term usage, according to interview with Dr. Eroshenko, who suggested this problem as well as solution, we designed 2 different kill switches in use in simple and complex circuits. This way, it can make the gene circuit have a much longer lifespan in case of deletion. In the meantime, we do acknowledge the downside of Cre-lox systems. Therefore we also would investigate into other systems including Hin recombinases. See the literature review for more details! Multiplexing How transient phenotype expresses knock-out model level metabolic engineering pathways? Many of the current metabolic engineering requires knock-out strains. For example, for some engineering is it essential to knock out the TCA cycle enzymes. While our model only expresses extra gene, and since phenotype need to change frequently, DNA knockout model is out of hand. Introducing: crRNA array with bacterial cmr or Cas13 system. In our design, we need fast and rapid regulation. Therefore regulation of the transcriptome would be a direct way. Phenotype 1 (B synthetic gene) Consider phenotype 1. the crRNA is able to 1. Downregulate native gene B 2. Downregulate phenotype II (A) gene Combining with expression of 1. Native gene C 2. Synthetic gene B Phenotype 1I (A synthetic gene) Consider phenotype 1. the crRNA is able to 1. Downregulate native gene A 2. Downregulate phenotype II (B) gene Combinng with expression of 1. Native gene C 2. Synthetic gene A What if the we engineer the crRNA to conditionally target the recombinase itself? It introduces a directionality of flipping. This could have multiple applications. One example would be to use alcohol resistant phenotype. For example, if ethanol level is low, both population survives in bioreactor. But if the ethanol level is high, the cells could have ethanol sensing module in the alcohol resistant phenotype, which activates the crRNA to downregulate Cre/other recombinase. This will allow the whole population to “shift” to the alcohol resistant phenotype without the killing of cells. The system is good since alcohol resistant phenotype may work slower in other pathways, but is more efficient towards the end of the fermentation. Further Multiplexing What if there are: 1. More than one promoter? 2. Promoter not at the side but at the middle, or within brackets? 3. No terminator so simultaneous 2 gene expression in circuit is viable? This will exponentially generate complexity in the gene design. Therefore -> check out dry lab, for a prototype database for circuits. Further Control systems 1. Level of recombinase expression The level of recombinase directly affects the ability of cell population to maintain ratio corresponding to metabolic and growth rate differences. 2. Ratio countering One of the ways to counter e.g. 2:7 growth rate in A:B phenotype, is to use a higher ratio in gene circuit, i.e. 1:3. To directly counteract the growth rate differences. This would allow less recombinase expression and flipping event. 3. Pulsing Since loss of function is a problem, pulsing by inducing temporally after some after in culture allows “reset” of the gene circuit to desired ratio. This also reduce the rate of deletion. However may not be necessary if using Hin recombinases. Limitation, Advantages, and Solutions Limitations According to our interview with Dr. Nikolai Eroshenko, also pointed out by our advisor and the HKUST team, the cre-lox system does not work well in bacteria. Moreover, even the tightest regulation system could not completely suppress the mobile DNA events mediated by recombinases. Moreover, loss of function by wild-type Cre is very frequent due to off-site targeting and recombination. Therefore, besides using the cre-mutants, we also adopted Dr. Eroshenko’s idea of incorporating kill switches. We will illustrate our design in the solution part below.. One other thing to consider is that our model now is using the multi-copy plasmid, but in lambda red system, the number of lox sites would decrease by 100 fold to several copies in one cell, due to single copy systems, therefore increasing the rate of reaction dozens of folds. Therefore, we also conducted experiments to see the effect of substrate copy number to the time to reach overall equilibrium. Please visit wet lab for the design and results. Advantages Comparing to the well-researched and the trend of using Quorum Sensing + kill/growth retardation switches, our system is less sensitive to external factors. Quorum sensing may not be applicable in many systems. According to a August 2020 Cell review paper written by Stephens et. al [2], QS systems is exciting field to develop, however still in early phase. And there exists problem relating to the real industrial application was explored in this QS system may not be applicable to most environments. This is also many reason why the industrial is unwilling to move from mono- cultures to QS mediated mixed culture, due to extensive optimization needed and innate complexity of the system. Since QS system also utilizes density of molecules, communication might be noisy. And in some projects like environmental remediations, threshold density may never be reached, rendering the system’s communication ineffective. Many systems also pair with kill switch/ growth retarding factors. This approach may decrease effeiciency, and also is very prone to mutations, due to extreme selective pressure posted by e.g. lysis circuits. Our system works without any communication module, it is only based on probability of distribution, and to regulate population, we do not use any growth limitators or toxins. Moreover, the recombinasion system works in mammalian, yeast, and bacterial cells. While QS system only works in microbes. Solutions By utilizing 2 approach of kill switches, we can elongate the circuit lifespan caused by deletion. Please also see model for simulations. Gene deletion causes lox to be cut out, activates the toxin expression. Originally blocked by 4 terminators inside the gene circuit. Gene deletion in other brackets causes loss of anti-toxin gene. This design is for circuit level >1, with >1 orthogonal lox sites. This also works if lox binds with other genome target, also causes loss of anti-toxin genes. For future Researches The recombinase system is orthogonal to other signaling systems within the cells. Instead of putting the whole gene cassette into the inversion complex, a transcription regulator that regulates a whole cassette of gene could be implemented. Although Cre-lox works in high gene distances, this approch allows shorter transcription products. Riboswitches could also be used together with the crRNA system to provide a low metabolic burden and fast regulations. Moreover, combining with other recombinases may offer more utility to the system, including serine recombinases which is famous for the gene memory and conditional logic gates (see literature review).
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HKU iGEM Team 2020


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