G.xylinus and E.coli belong to the Gram-negative bacteria, and G.xylinus can secrete bacterial cellulose,which has great potential in many areas. In order to reach the diferent demand of nutrition and carbon source, we have finnished exploring the better growing together environment for both bacterials. And then, we have made G.xylinus and E.coli growing in the Co-culture media together and get BC membrane after a week inoculated.
Key Achievements
Create the Co-culture system.
Find a better inoculation ratio and carbon source.
Testing all of the parts working and get BC membrane datas.
Create a response surface modelling in the Co-culture system.
Improvement :
Optimized co-culture media can increase the yield of BC by16.7%
Optimized culture media can shorten the time for G.xylinus to produce the same amount of BC than the original one.
G.xylinus
Overview :
The biosynthesis pathway of bacterial cellulose in Gluconacetobacter xylinus is complicated including the synthesis of cellulose precursor, polymerization of glucan monomers and assembly of glucan chains.
Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering,and the yield of BC's is also low. According to our finding that the yield of BC was higher than that of G.xylinus cultured individually when cultured with E.coli at pH=5.We have overcome the numerous difficulties associated with G.xylinus genetic engineering, and we mainly do the following two aspects of work :
overexpress key gene of PGM：ptimize the metabolic pathway of Gluconacetobacter xylinus., redirecting more carbon sources into the production of bacterial cellulose
Overexpress key gene of c-di-GMP: increase the production of cellulose synthase.
As a result, the production of BC produced by G.xylinus CGMCC 2955 strains have dramatically improved ，and we made an important contribution to improveing BC production.
Key Achievements
Overexpressed key gene of PGM ：researched the effect of PGM on the yield of BC.
Overexpress key gene of c-di-GMP：researched the effect of increasing the production of
cellulose synthase on the yield of BC,the experimental result is positive.
Constructed 6 kinds of vectors which can express both in G.xylinus and
E.coli，
Compared the effects of different promoters -PAsr and J23119 linked with PGM on the yield of
BC.
Compared the effects of different promoters -PAsr and J23119 linked with c-di-GMP on the yield
of BC.
The constructed G.xylinus and E.coli were cultured under co-culture conditions.
Having obtained a new construction of the vector Gluconacetobacter xylinus strains can effectively improve the yield of BC .
Improvement :
G.xylinus containing plasmid BBa_K2267005 have a higher yield of bacterial
cellulose. The data ...
G.xylinus containing plasmid BBa_K2267006 have a higher yield of bacterial cellulose. The data ...
E.coli
Overview :
Escherichia coli is the organism of choice for synthetic biology. It as a model strain, has the character of clear genetic background, technical operation convenient, simple conditions of cultivation.
As the G.xylinus in the fermentation process of glucuronic acid and other acidic substances, pH declining, can not be the best pH value of the conditions of growth, the introduction of E.coli, E.coli production of alkaline substances, and the transformation of E.coli and G.xylinus Co-culture, to create co-culture system, to achieve the stability of E.coli pH value of the environment, so that the purpose of G. xy high yield.
Key Achievements
Make GlsA working. GlsA encodes an acid-activated glutaminase, which is sufficient for an acid resistance system, and it is able to catalyze a reaction converting glutamate to glutamine and releasing the free gaseous ammonia.
Make Pasr connect with GlsA. Asr promoter，Pasr， belongs to asr gene encoding acid-shock RNA. It is a pH-responsive promoter native to E.coli which induces transcription in acidic environment from pH 4.0 ~ 5.0 , and shows low activity at a neutral pH. We chose asr promoter as the base of pH regulation system.
After the connection of Pasr and GlsA, it was introduced into Escherichia coli to stabilize it at about pH = 6, and co-cultured with Acetobacter xylophilus at the optimum pH to produce high-yield bacterial cellulose.
The mutation parts
Overview :
In the world of synthesis biology,the prompters’ sensitivity always determine the rate of parts or systems reactions and mutation is a kind of the usual method to improve parts. So we designed 13 groups of point mutations around the -10 areas to improve the sensibility of the promoter. And then, we used the GFP protein to instead of our functional genes when we tested the mutation results.
Key Achievements:
We have already designed 13 groups of the point mtation to improve the sensibility
of the promoter and finished the most of mutation works.
Comparing with the target sequeces, the piont mutation sites are in line with our
expectations.
In summary, we have obtained 5 groups of the successful point muations which performance can improve a lot campared with the nature promoters.
Improvement :
Modified E.coli with plasmid BBa_K2267001 can stabilize the pH in the culture
media.
Via point mutation to plux, these promoters have higher efficiency than the original plux.
Bronze Medal
✔ Register the team
✔ All deliverables were met!
✔ Our attributions page credits those who've helped us along the way!
