Engineering
Welcome to the engineering page!
The main goal of CosMic is to create a self sustainable bioink produced by Azotobacter vinelandii. The ink will consist of two biopolymers, cellulose and alginate, which are both synthesised by our host organism. Alginate works as the base of the bioink and cellulose ensures stability. Alginate is naturally produced by A.vinelandii, so our goal is to overexpress the corresponding gene to obtain a higher alginate yield. In order to produce cellulose in A. vinelandii, the cellulose operon from Komagataeibacter xylinus is transformed into our host organism. Here we describe the engineering cycle of our project, what challenges we faced and how we tried to overcome them.
Alginate
Design
The goal of CosMic is to get the highest possible alginate yield in order to create the base of our ink. We chose Azotobacter vinelandii since it already provides a high yield of alginate (5,8 g/l). The overexpression of alg8 increases the alginate production in our organism. Alg8 codes for the glycosyltransferase in the alginate operon and is located in the inner membrane of A. vinelandii. It polymerizes alginate precursors such as GDP-mannuronic acid and guluronic acid1. In this part of the engineering cycle, we did not only design our construct, but we also thought of methods to solve our problems. During our project we often came back to this step to define our problem, rethink our methods and try a different approach.
Build
In order to overexpress alg8 the gene was cloned into a high copy plasmid (pSB1C3) along with a His-Tag and an arabinose Promoter, as you can see in the figure below. We used the arabinose promoter, because Prof. Dr. Oliver Einsle, director of the Institute of Biochemistry in Freiburg, recommended it. The overexpression is linked to the arabinose promoter, since it controls the expression through the arabinose in the media of the bacteria. This also allows us to estimate the metabolic load in A. vinelandii of the modification. In addition to this, the protein yield can be determined on the basis of the His-tag.
Test
At first we tried to amplify the vector, the arabinose promoter and the gene block alg8. The amplification of the promoter worked great, but we had several problems with amplifying the vector and alg8. The vector amplification product did not align in the gelelectrophoresis as expected, and the band of alg8 just appeared as a smear and was not clearly identifiable.
gelelectrophoresis with our alginate construct components from several approaches
At first we tried to solve this problem by changing our method to a gradient PCR. Then we tried a different mastermix by adding MgCl2. Unfortunately, we still could not identify the correct fragments. That is why we went back to the design step of the engineering cycle and designed a different set of primers. We focused on the alg8 gene block, since the promoter showed good results. The primers were completely new designed, because the melting temperature was adjusted for a new polymerase. We fixed the melting temperature and removed the overhangs to detect the effect on the results of our PCRs. After many trials, we could observe that we could not improve, and we got the similar results like we had at the beginning. Lastly, we tried to split the amplification of the alg8 gene block into two parts. For that, we designed new primers once more. Now we could see the band more clearly, but we could not specify the respective parts of the split alg8 block, because we suspect that issues during the gelelectrophoresis occured.
alg8 PCR products with different primersets from 27-Sep.2022
Unfortunately, due to time constrictions, we were not able to repeat this PCR to get better results. With more time, several more repetitions would have been possible. As a consequence, we could have gained a better learning why the amplification of the alg8 gene block did not work well.
Learn
The main challenge of the alginate engineering cycle was the amplification of the alg8 gene block and optimising the PCR conditions. We tried to solve this problem by changing our methods (e.g. doing a gradient PCR), designing different sets of primers (with or without overhangs), trying a different mastermix (adding MgCl2) and different polymerases. We went through many iterations of this cycle since we tried a lot of different approaches for problem-solving, by repeating the initial step of the engineering cycle and testing it. Even without perfect results, an exclution of various sources of error were possible.
Cellulose
Design
The bioink of CosMic consists not only of alginate, but also of cellulose. Cellulose provides stability in our manufactured products. To make A. vinelandii produce cellulose, it was nessesary to transform the cellulose operon from Komagataeibacter xylinus into our organism. We chose this organism, because its cellulose not only shows astonishing mechanical strength, but also is the most efficient cellulose producer among the other bacterial cellulose producers2.
Build
First we tried to design our own construct with all four catalytic subunits AcsABCD and three ancillary proteins cmcax, ccpAx and bglxA, but we faced a lot of challenges. The main problem of the construct was its size (>10kb) and its high GC content (>60%), which made it really difficult to synthesise. We tried to narrow it down to just the catalytic subunits AcsABCD, but the problems could not be solved by doing so. We wanted to build the cellulose operon from the parts that could be optained from the distribution Kit, made by the iGEM-team Imperial (2014). In order to test the operon, we wanted to transform it in Escherichia coli, later in Azotobacter vinelandii. By this we could test, if the cellulose operon would work in another organism. We designed primers for the parts and started with the transformation.
Test
The first step was to optain cellulose parts from the distribution Kit, made by the iGEM-team Imperial (2014), by tansforming them into E. coli. We used the catalytic subunits AcsAB (BBa_K1321334) and AcsCD (BBa_K1321335) and tried to transform them into competent E. coli cells. Just like with the PCR of the alginate construct, we faced a few challenges with the transformation of the parts. Unfortunately, the transformation did not work several times. So we tested the parts from different distribution kits (2019 and 2021) to see if the problem was a mistake in the implementation or if something was wrong with the distribution kit, like wrong storage or transportation. As we still did not get any good results, we tried a different protocol and changed the parameters of the transformation by raising the incubation time, the temperature and duration of the heat shock and the amount of DNA to transform. This troubleshooting process was succsessfull so that growing colonies on the plate with the catalytic subunits AcsCD were visable. We miniprepped the plasmid and stored them. Unfortunately, it did not work for the catalytic subunits AcsAB and due to time constriction, we only reached the initial part of our goal to synthesise cellulose in E.coli.
Learn
If we had transformed the catalytic subunits AcsAB, we would have assembled both parts with the arabinose promoter through Gibson cloning. This part of the project was very promising, since we achieved a successful transformation and could have lead to good results.
- Urtuvia V. et al., Bacterial alginate production: an overview of its biosynthesis and potential industrial production (2017), World J Microbiol Biotechnology
- Brown RM Jr, Willison JH, Richardson CL. Cellulose biosynthesis in Acetobacter xylinum: visualization of the site of synthesis and direct measurement of the in vivo process. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4565-9. doi: 10.1073/pnas.73.12.4565. PMID: 1070005; PMCID: PMC431544.