Team:BIT-China/Description
Inspiration
We might not have a casual spark of inspiration, but we do have a strong sense of mission which bound us to the great industry of production.
Our project was born in history. Looking back on BIT-China's history of iGEM, projects are always closely related to industrial production. The predecessors have put forward their own intelligent cell factories scheme in various aspects of microbial industrial production.
The construction methods of traditional cell factories mainly include the design and optimization of metabolic pathway, the regulation and optimization of gene expression, the optimization of tolerance and the systematic improvement of production conditions. Based on traditional cell factories, the intelligent cell factories can respond to the changes of environment and its own state independently and adjust the environment or production state independently.
In terms of production technology, the intelligent cell factory built in 2013 can respond to temperature (I'M HeRE, 2013), the intelligent cell factory built in 2015 can respond to pH (pH controller, 2015), and the system built in 2018 can respond to redox state (who can get an A, 2018). They have realized the intelligent recognition of the key environmental factors such as temperature, pH and redox in the fermentation process. And then the cell factories can regulate the environment and have higher tolerance, which saves energy, reduces emissions and increases production.
In terms of production safety, the project in 2014 guaranteed the safety of production technology in cell factories and ensured that industrial strains would not outflow (E.co-Lock, 2014).
In terms of production efficiency, the project in 2016 can give cell factories the ability to intelligently remove the "lazy" cells that have lost plasmids in the population, and improve the production capacity of the population (P-SLACKiller, 2016).
Looking back into the history, the construction of a new intelligent cell factory has the same origin as BIT-China.
Looking forward into the future, an era of intelligent cell factories is in our vision.
Now, we hope that engineered bacteria can recognize their own growth state, manage resources intelligently and know when to produce and when to grow.
Description
Constructing bio-synthetic pathways in engineering microbial cells to produce fine chemicals has been considered as a sustainable, environment-friendly way in modern chemical production. However, these exogenous synthetic pathways directly compete with endogenous metabolic pathway in the usage of both metabolites and “transcriptional resources”. Moreover, over-expression of enzymes in exogenous pathways and synthesis of toxic intermediates would cause growth retardation of these microbial cells. It means that the ideal state for chemical production often conflicts with cell growth.
Obviously, we need strategies to manage the relationship between growth and production. Ideas of balancing cell growth and production processes long exist. Traditional ways to achieve this involve using inducers to initiate product synthesis, which is time-consuming and expensive for large-scale production. For more economically viable industrial usage, we develop self-induced system to control the transcription of exogenous genes, naming it “Achieved Transcription Management”.
The system allocates intracellular “transcriptional resources” to growth-related genes in early stages of fermentation and switches them to product-related genes after a high cell density is achieved. Under the guide of mathematical models, the proper cell density is responded by quorum sensing system turning on downstream genes, including σ factors and T7 RNA polymerase. And they control the transcription of exogenous genes responsible for lycopene production.
In addition, modeling and calculation of enzyme activities help decide the quantity of enzymes most suitable for reducing the pressure of enzyme synthesis on cells.
We classify transcriptional resources into two categories: one acts as substrates in transcription, such as nucleotides, and the other assists in the process of transcription, such as RNA polymerase. Preferably, we call the first one building blocks and the second builders.
To manage the first type, we use T7 expression system to achieve the demand-based distribution of nucleotides with highly substrate-competitive, exogenous T7 RNA polymerases, a process resembling engineers gathering building blocks.
As for the second, we find σ factor orthogonal expression system very effective to recruit our builders— RNA polymerases— according to demand.
We compare the production and the population density of our engineered bacteria to those of the bacteria that do not have the system after cultivating in the same culture during a equal period of time. Lycopene, a colored and commercially valuable compound, is selected, in this case, as a product of purpose to test whether if our design truly balances growth and production.