Optimization of Fermentation Conditions
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
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
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).
- 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
The Establishment of Co-Culture System
Based on what we have illustrated above, it is our priority to both improve the yields of cellulose generated from G.xy though exploring and optimizing the various condition of co-culture system during the fermentation process and make sure “engineered bacterial”——E.coli have grown stably and normally.
Since the reproduction speed of E.coli have been much faster than the speed of G.xy, it is significant to calculate the ratio of inoculating both bacterial. Yields of cellulose will be compromised if we inoculate a number of E.coli while little volume of E.coli for which will fail to manipulate the pH.Therefore, it is necessary for us to to explore the best ratio of incubating E.coli and G.xy.
During the process that we utilized culture medium for G.xy to fermented both E.coli and G.xy, the speed of forming the biofilm has been slowed down, therefore we went through the study of growth and metabolism of G.xy and noticed that the glycerol has played a crucial role in metabolic route of G.xy. According to the researches, adequate amount of glycerol is able to stimulate G.xy to secrete BC in the later phase. Based on what we have in mind, we decided to adjust the combination of carbon source of culture medium, which is turn the former pattern that glucose as the single carbon source to integrated carbon source pattern which use the glucose and glycerol to co-provide carbon source.
In addition, we as well as did the trails to measure the impact of yields of BC from other impact factor as ethanol and citric acid etc..(Glu/Gly = Glucose/Glycerin)
We designed the table below and followed the steps to process our experiments, expiring different ratio of incubation and carbon source. Moreover, we dried the BC from day 7 culture medium and measured both the weight and thickness to obtain relative data.
The comparison of weight and thickness of BC showed that on the condition of equivalent carbon source and inoculated ratio of G.xy and E.coli is 4/1, the yields of BC will at least advance 11.5% compared to other groups. At the same time, we as well as discovered that the capacity of G.xy secrete BC has been significantly improved with the ratio of glucose and glycerol is 3/2~4/1.
After setting up the inoculation level and the range of carbon ratio, we moved forward to optimize the carbon source through RSM. During this process, we collected a series of data which based on the yield as main target and the pH residue as co-factor, the data is present below:
The finally optimize formula which has already reached the ideal ultimate pH and visibly improved the yields of BC is :
formula Glu(g) Gly(ml) 50%EtOH Predicting Final pH Target 15.11 9.53 0.05 4.36114
this optimized formula has similar carbon ratio to what we have measured in the culture medium of previous experiments, therefore we ultimately set up the carbon ratio as glucose/glycerol=3/2 of our co-culture medium.
There is what we have gathered after utilized 3/2 medium in the co-culture system based on the former experiments:
Compared the pH residue of single G.xy culture and co-culture during the fermentation, obviously approximately 15h after inoculation, pH of co-culture started to significantly drop while the yields of BC from co-culture clearly outnumber the yields of single G.xy culture.
- Factors affecting the yield and properties of bacterial cellulose
- Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions
- Dynamic control and quantification of bacterial population dynamics in droplets
Optimization of metabolic pathway
Cellulose can be synthesized by many organisms, ranging from prokaryote to animal. In prokaryotic organisms, bacterial cellulose is synthesized mainly by Gluconacetobacter, Rhizobium, Agrobacterium, Escherichia, Enterobacter and salmonella. In nature, only bacteria can synthesize cellulose pellicle with water-holding texture, among which the most studied bacteria are the genus Gluconacetobacter. Several common strains in the genus Gluconacetobacter can be distinguished by different representations. Bacteria that can synthesize bacterial cellulose are mainly G. Europaeus, G. intermedius, G. Oboediens, G. Nataicola, G. Sacchari and G.xylinus. Researchers have been studying the biosynthesis of bacterial cellulose in recent decades. This G.xylinus has long been recognized as a model strain. Many classical cellulose synthesis pathways and regulatory models were obtained in G.xylinus. Therefore,we choose G.xylinus as the research object in this experiment.
The biosynthesis pathway of bacterial cellulose in G.xylinus is complicated including the synthesis of cellulose precursor, polymerization of glucan monomers and assembly of glucan chains.
The biosynthesis of bacterial cellulose begins with the conversion of substrates to cellulose precursors, which are usually carbon sources, such as the conversion of glucose to uridine diphosphate glucose (udp-glucose). Uridine diphosphate glucose is the substrate of cellulose synthase, and it is catalyzed by cellulose synthase to form β-1 and 4 glucoside bonds between glucose monomers. There are specific sites on the outer membranes of the bacterial cells, which are coupled with the cellulose synthase complexes and is the spot where the polymerization is conducted . When the polymerization reaction is over, the newly generated cellulose is secreted to the extracellular through these sites. The following figure is a partial schematic of the production of cellulose by G. xylinus
We hope to optimize the metabolic pathway of bacterial cellulose, redirecting more carbon sources into the production of bacterial cellulose; meanwhile expressing key genes in BC synthesis process. Through these two methods we hope to achieve high yield of BC . Therefore, we designed the following experiments:
Design & Engineering :
Phosphoglucomutase(PGM) is able to catalyze the conversion of glucose-1-phosphate and glucose-6-phosphate and plays an significant role in both glucose metabolism and BC metabolic network.
Cyclic dimeric guanosine monophosphate (c-di-GMP) is a new type of second messenger molecule that is ubiquitous in bacteria. Bacteria utilize it to perceive cell surface signals and activate targets within cells, which involves amplification of the original signal and the subsequent expression of a series of specific genes in the cell. These genes affects the various physiological and biochemical processes in the cells. c-di-GMP control a wide range of process, and has a role in transcription, translation and post-translation levels. In addition, c-di-GMP participates in the regulation of multiple biological functions and affects biofilm formation. In a nutshell, it is a crucial elements to the formation of membrane-like bacterial cellulose and the improvement of BC production.
This experiment attempts to improve BC production by over-expressing PGM and c-di-GMP, a small signal transfer molecule of bacteria.the goal is to implement both to realize the high yield of BC.
The process of the experiment is divided into following steps, firstly, we transform the constructed plasmids into the E. coli DH5α. When expressing successfully, then we transform it into G.xylinus through electroporation and put it in comparison with the common G.xylinus. We try to explore the influence of each plasmids on the production of BC after fermentation.
We use PCR to amplify gene PGM and c-di-GMP，and transform reconstructed plasmids J23119+PGM and J23119+c-di-GMP into G.xylinus. Then we put them in comparison with the control group PAsr+PGM and PAsr+c-di-GMP and measure its effect on the yield of BC respectively.
At the beginning of the experiment, it was found that the yield of BC was higher than that of G.xylinus cultured individually when cultured with E.coli at pH=5 ，so we first construct the PPGM-PGM-331 plasmid and verify its effect on BC yield after overexpression.
The results show that the overexpression of PGM has no significant effect on the increase of BC yield.
We acquire genes of PAsr from K12 genome of E.coli via PCR
We acquire genes of PGM and c-di-GMP from genome of G.xylinus.(With and without PGM)
Construction of vector PPGM-PGM-pSEVA331. Transformation of plasmids into E.coli.
Verification of homologous recombination via colony PCR
Co-culture of E.coli and G.xylinus
Second, we found a strong promoter -J23119, linked it with the PGM, at the same time linked it with c-di-GMP which is a signal molecule could improve the yield of cellulose synthase.
The results show that the yield of BC was not significantly increased, but the yield is increased compared to PPGM-PGM-331 plasmid, specially J23119-c-di-GMP.
- We acquire gene sequence J23119 and complete its linking with PGM and c-di-GMP
- We insert gene segments into plasmid pSEVA331 and transform it into chemically-competent E.coli.
- Verification of homologous recombination via colony PCR
Co-culture of E.coli and G.xylinus
Third, due to G.xylinus is co-cultured with E.coli in the environment of pH=5 as we designed, we also try to linked PGM with the acid-controlled promoter PAsr, and linked c-di-GMP with PAsr. We explore and compare the effects of overexpressing PGM and c-di-GMP on the yield of BC in the environment of co-culture and pH=5. At the same time,the effect of it and pH-responsive promoter PAsr on BC production will be compared.
The results showed that the yield of BC was further improved than that of the second step, especially when PAsr was connected with c-di-GMP, and the yield of BC was significantly improved under mixed culture.
- We acquire gene sequence of PAsr＋PGM via PCR
- We acquire gene sequence of PAsr＋c-di-GMP via PCR
- We insert gene sequences into plasmid pSEVA331and transform them into chemically-competent cell into E.coli.
- Verification of homologous recombination via colony PCR
Co-culture of E.coli and G.xylinus
- Peter Ross， Raphael Mayer， and Moshe Benziman. Cellulose Biosynthesis and Function in Bacteria [J] . Microbiological Reviews，1991，55(1):35-58
- Peter Ross, Raphael Mayer, Haim Weinhouse, et al. The Cyclic Diguanylic Acid Regulatory System of Cellulose Synthesis in Acetobacter xylinum[J]. The J. of Biological Chemistry, 1990,265(31):18933-18943
- Alan L. Chang, Jason R. Tuckerman, Gonzalo Gonzalez, et al. Phosphodiesterase A1, a Regulator of Cellulose Synthesis in Acetobacter xylinum, Is a Heme-Based Sensor[J].Biochemistry, 2001,40:3420-3426
BC Property Testing
After the experiments above, we processed the bacterial cellulose film formed by the co-culture of the modified E. coli and G.xylinus, we would like to know whether this culture method of mixed bacteria will change the microstructure of BC? Therefore, we observed the morphological characteristics of the bacterial cellulose produced by the co-culture of the bacteria by scanning electron microscopy (SEM), infrared spectroscopy (FTIR) and atomic force. The results are as below: :
1. Scanning electron microscopy (SEM)
We first used a scanning electron microscope to observe the formation of bacterial cellulose samples under different electric field strength. The results are shown in the figure
The microstructure, fiber detail and fiber width of the bacterial cellulose samples can be analyzed by scanning electron microscopy after freeze-drying. We found no significant difference between them.
2.Infrared Spectroscopy (FTIR)
The use of Fourier transform infrared spectroscopy can help us understand the chemical structure of cellulose. Bacterial cellulose has an absorption peak for a particular wavelength of infrared, so that the composition of its chemical group can be analyzed.
Respectively, we analyzed BC film under the two conditions after the natural drying by the infrared spectrum scan, the results shown in Figure. The absorption peak at about 1060 cm-1 reflects the vibration of the C-O-C group, which is the characteristic absorption peak of the cellulose. The absorption peak at 1428 cm-1 is caused by the bending vibration of the C-H bond, and the depth is related to the crystallinity of the cellulose. Although the infrared spectrum is a simple method, it can only give a relative value, it can show whether is a crystalline phase or non-crystalline phase at one time. It can be seen that the BC film produced under these two conditions are not much different in the composition of the structure.
3. For the quality of wound dressings
Bacterial cellulose is well suited for wound dressings due to its good biocompatibility, high mechanical strength in wet state, good liquid and gas permeability, and inhibition of skin infections. For secondary and tertiary burns, ulcers have been successfully used as temporary substitutes for artificial skin. For the application of bacterial cellulose in medical materials, our team visited the BC factory and learned more about what conditions are required for the products of BC films used as wound dressings.
The above test content：
Test Items Technical Requirements Clause Technical Requirements Test Result Conclusion Water Content 2.2.3 ≦5 5 qualified pH 2.2.5 6.0±2.0 6.3 qualified Heavy Metals 2.2.6 ≦10 <10 qualified Residue 2.2.7 <4.0 0.3 qualified
At the same time we would like to thank xx company for providing the conditions of the test of our BC films . After a variety of tests of specifications, size, permeability, water content and others, data shows that the bacterial cellulose produced after the co-culture meet the technical requirements of wound dressings.
During the experiments of overexpressing PGM, we discovered that it didn’t increase the yield of BC as expected. After researching relevant literature,we assume that due to the fact that G6P plays a significant role in the whole metabolic network, over expressing PGM may affect the direction of energy flux in the network. Therefore, the energy in the route of producing BC is going down leading to no increase in the yield of BC. On the other hand, perhaps the reaction PGM catalyzed is not a rate-limiting step or the overexpression of PGM hasn’t improve the efficiency of the enzymatic reaction. Other then that, it is very possible that there are still factors that we haven’t covered influencing the whole metabolic process and the yields of BC.
Therefore.in the future, we might consider to modify or overexpress UGP which catalyze a rate-limiting reaction and explore its effect on the yields of BC
Bacterial cellulose is already a useful biomaterial as is due its attractive mechanical properties. So we want to make and characterise new cellulose binding domains, and fuse them to functional proteins. In our project, the bingding domains are the spytag and its catcher parts--spycatcher. We have designed a new spytag, including its RBS region, and make spytag link with GFP protein genes. We would verificate the catching power about this parts and do more experiments to give more functional properties into the bacterial cellulose.
As we have increase the yield of bacteria cellulose fulfiling the protection of the membrance will wake the BC functional Since the BC membrance is accepted due to its mechanical properties，we would like to introduce another system to add new characteries to our product. According to the project of team Peking 2016 and resegrd papers we want to introduce the spytag-spychther system which is commonly used for binding. labeling immobilization and creating new kinds of application. We culture G.xylinus with the E.coli which can produce small peptid in the culture media evenly；thus， the spycather can be inlaid with BC membrance. The functional protein can be fused with spytag and be expressed bu the E.coli eddicieatly. We fuse the spytag with GFP.