Team:TUST China/Ecoli
Optimization of
Fermentation Conditions
Optimal pH
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During the fermentation process, problems like low efficiency of gene expression and low yield always occur. The formula of culture media and the condition of culturing in fermentation have direct or indirect relations with these issues. Particularly, pH can affect the dissociation of some certain components and intermediate metabolites ，which can influence the capacity utilizing these materials of bacteria. Meanwhile, the fluctuant environmental pH have certain impact on the pH level in the cells in the form of weak acid and alkali influencing the metabolic reaction of the cell. Therefore, an appropriate control of pH is crucial to the effective gene expression. That is the reason why we need to discover the optimal pH for the fermentation of G.xylinus. The scope of pH variation in the culture media during fermentation process is wide. In order to study the optimal pH for G.xylinus fermentation, we use acetic acid with 200 ionic strength to maintain the pH in the outside environment.
The scope of pH variation in the culture media during fermentation process is huge. In order to study the optimal pH for G.xylinus fermentation, we use acetic acid with 200 ionic strength to maintain the pH value in the outside environment.
Initial pH Buffer pH after ten days of fermentation pH of the control group after ten days of fermentation 6.25 5.81 5.83 5.88 4.1 4.07 4.07 5.75 5.58 5.64 5.45 4.06 4.1 4.06 5.25 5.3 5.3 5.26 4.04 3.95 4.03 4.75 4.84 4.83 4.76 3.99 3.88 3.98 4.25 4.02 4 4.01 3.82 3.81 3.81 
As you can see on the graph, the culture media with buffer in them has low pH variation. We have processed the membrane collected after 10 days of fermentation and the table below is the data we have collected.
As the statistics show, when pH=5.75～5.25 ,the yield of BC the most optimal.Meanwhile, when pH=5.25 and 4.75, the yield of BC are higher than the control group by 11% and 7%.
Therefore, we hope we can maintain the pH at 5 in the environment during G.xylinus fermentation.
Transformation of E.coli
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Due to the production of acidic substances like glucuronic acid during G.xylinus fermentation, the accumulation of acidic compounds lead to the decrease in pH. In order to maintain the optimal pH for producing bacterial cellulose, a mechanism to produce alkali substances is required. We choose model strain E.coli considering its benefits such as having a clear genetic background, easy technical operation and short culture time. Therefore,we plan to modify E.coli to have the ability to respond to the variation of pH and enable it to produce alkali substances when the environment is acidic enough. Hence, the alkali substances produced can maintain the optimal pH for G.xylinus to produce BC.
First, we need to find a switch with the function of sensing the pH level in the environment and respond accordingly. Luckily, we have identified a proton type of promoter P-asr located on the activator protein binding domain. When pH is around 5, it can activate RNA polymerase to combine with template DNA precisely resulting in the expression of downstream sequence. The pH for the activation of P-asr is very close to the optimal pH for producing BC. Therefore, we decide to use P-asr as our switch.
After that, we have identified a gene GlsA with the function of producing alkali substances via the expression of glutaminase. This enzyme can catalyse the conversion of glutamine and water to glutamate and ammonia. The ammonia will combine with H+ to increase the pH in the environment.
We have verified its capability of producing alkali in DH5α
Based on what we have acessed, we know that GlsA is activated when the pH is below 6. The graph above also demonstrates that the pH variation in the culture media of E.coli also fluctuate around 6.
We add the gene of P-asr before the gene of glsa and construct a plasmid with pet28 as the backbone. We then transform it into E.coli DH5α and test the variation of pH during fermentation. The results is presented by the line graph below and we put it in comparison with the one with empty plasmid and the one with only GlsA. We can observe that the pH is maintaining at around 5.
We put the transformed E.coli in co-culture with the transformed G.xylinus, detected the BC film they produced. The data are as follows.
Control of Population Level
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We worry that the if the cell concentration of E.coli is too high, E.coli will compete with G.xylinus for nutrients leading to an negative impact on its growth. Therefore, we introduce Quorum sensing system and autophagy gene φx174E in E.coli.
In order to control population levels , we engineered a synchronized lysis circuit (SLC) using cou- pled positive and negative feedback loops that have previously been used to generate robust oscillatory dynamics. The circuit(BBa_K2267040) consists of a common promoter that drives expression of both its own activator (positive feedback) and a lysis gene (negative feedback).
Specifically, the luxI promoter regulates production of autoinducer (AHL), which binds LuxR and enables it to transcriptionally activate the promoter. Negative feedback arises from cell death that is triggered by a bacteriophage lysis gene (φX174 E) which is also under control of the luxI promoter. AHL can diffuse to neighbouring cells and thus provides an intercellular synchronization mechanism.
Quorum-sensing bacteria produce and release chemical signal molecules termed autoinducers whose external concentration increases as a function of increasing cell-population density.
Bacteria detect the accumulation of a minimal threshold stimulatory concentration of these autoinducers and alter gene expression, and therefore behavior, in response. Using these signal-response systems, bacteria synchronize particular behaviors on a population-wide scale and thus function as multicellular organisms.
We modify the plux hoping to increase its sensitivity and we choose to conduct point mutation in the region of -10 according to the literature. Down below is the exact mutaion point and we add an indicator gfp after plux. We try to acquire a plux with higher sensitivity via modelling.
The bacterial population dynamics arising from the synchronized lysis circuit can be conceptualized as a slow build-up of the signalling molecule (AHL) to a threshold level, followed by a lysis event that rapidly prunes the population 。 After lysis, a small number of remaining bacteria begin to produce AHL anew, allowing the ‘integrate and fire’ process to be repeated in a cyclical fashion. We observe growth with the fluorescent protein superfolder GFP (sfGFP).
Reference
- A Glutamate-Dependent Acid Resistance Gene in Escherichia coli
- Functional and Structural Characterization of Four Glutaminases from Escherichia coli and Bacillus subtilis
- Glutaminase of Escherichia coli
- Rational design of an ultrasensitive quorum-sensing switch
- Genetic programs constructed from layered logic gates in single cells
