Results
Goal: Overexpression of alg8 in Azotobacter vinelandii
Results: For overexpression of alg8 in Azotobacter vinelandii we first tried to amplify the alginate construct consisting of the vector pSBC13, alg8 and the arabinose promoter. The amplification of the arabinose promoter worked very well, but we had many problems with the amplification of the alg8 gene block and the vector pSBC13. The vector could not be amplified correctly and alg8 just appeared as a smear and was not clearly identifiable. We tried to solve our problems by using different polymerases (Q5 and Taq Polymerase), designing different sets of primers, with and without overhangs, and changing our methods (e.g doing a gradient PCR). With a lot of effort and well thought adjustments we could not fulfil our goal of amplifying the alginate construct and overexpressing it in Azotobacter vinelandii. Please visit our Engineering cycle page if you want to know more about it.
gelelectrophoresis with our alginate construct components from several approaches
Future Direction: With the help of our failed attempts, future iGEM-teams are more able to estimate the source of failed PCRs and methods of problem solving.
Goal: Transformation of the cellulose operon in Escherichia coli
Results: In order to transform the cellulose operon from the organism Komagataeibacter xylinus into Escherichia coli we used the parts from the distribution kit. We tried to transform the four catalytic subunits AcsAB (BBa_K1321334) and AcsCD (BBa_K1321335) from the distribution kit of 2021 and 2019. It did not work several times with the parts from both kits and therefore we concluded that there must be a problem with the implementation. That is why we tried a different protocol and changed the parameters of the transformation by increasing the amount of DNA to be transformed, raising the incubation time and the duration of the heat shock. Fortunately, the transformation worked for the subunits AcsCD, but not for the subunits AcsAB. We mini prepped the plasmid and stored it for future usage.
Future Direction: For the future our data can be useful for clarification and optimising the process of transformation, especially the single tube variation.
Goal: Modelling Azotobacter vinelandii metabolic pathways using FBA (flux balance analysis)
Results: We were able to quantify the sucrose intake in relation to Alginate production. In general, we used our inoculated A. vinelandii for different kinds of experiments to form a data based flux. The most essential data, like cell density and dry cell mass, needed to be correlated to create a visual model. Also the sucrose intake, which we measured with a sucrose kit from abcam (ab83387) and also the alginate production were significant information to our model. If you want to know more about our model, visit our modelling page
Future Direction: Making it easier for future iGEM-teams to work with A.vinelandii.
Goal: Build a bioprinter and reach perfect consistency for our bioink
Results: To bring our project to life, we converted an Anet 3D-Printer into a bioprinter by printing the needed parts ourselves and buying a new motor. A big part of the conversion is the new software we added to adjust the movements for a Bioprinter. Additionally, the consistency of our BioInk has had to be adapted to our printer, so we tried it with commercial alginate and cellulose and their composition together. Visit the printer contribution for more information.
Future Direction: We developed tips and tricks to build your own bioprinter for future iGEM-teams.