Team:Chalmers Gothenburg/Results
Construct assembly and modifications of organisms
There were many planned modifications for each of the 5 organisms that were going to be used. These were supposed to enable the production organisms for the dependency system of the co-culture and to increase the acetate production of Synechocystis. However, throughout the project there were issues with getting the desired constructs assembled from various PCR products. Techniques such as Gibson assembly and Fusion PCR were employed varying success. It was thus not possible to carry out many of the desired modifications in the time frame dedicated for the lab work.
Which constructs were successfully assembled can be seen in Table 1.
| Synechocystis constructs | Assembled? |
|---|---|
| Knock-out argH + insert ackA | No |
| Knock-out glnA + insert AckA | No |
| Knock-out Acs + insert RFP | No |
| Knock-out Acs + insert pta | No |
| Knock-out argH | No |
| Knock-out glnA | Yes |
| Knock-out acs | Yes |
| E. coli constructs | Assembled? |
| Overexpression of point mutated argA + GFP insertion | No |
| ArgR knock-out | No |
| B. subtilis constructs | Assembled? |
| Glyoxylate shunt | Yes |
| ahrC knock-out | No |
| Y. lipolytica constructs | Assembled? |
| Overexpression of gln1 and GFP | No |
| Overexpression aqr1 | Yes |
The reason S. cerevisiae is absent from Table 1 is that the work for that organism was primarily focused on the promoter study and promoter exchange at first, the results of which then were needed to plan further constructs. Although the promoter study was completed, the continued work on S. cerevisiae was put on hold for other tasks that at the time were deemed more important.
Synechocystis growth trials
A growth trial was performed on both the WT and JA06 strains of Synechocystis given to us to study its growth rate, Figure 1, and the acetate production, Figure 2 & Figure 3. As can be seen in Figure 1 the wild type and mutant strain JA06 of Synechocystis followed a very similar growth curve during the 600 hours long growth trial. This is good since it indicates that the added features of the mutant strain does not put any major metabolic burden on the organism.
When measuring the samples from the growth trial by using HPLC it was shown that the wild type had no acetate production, as was expected. The JA06 strain showed acetate levels of up to 0.22 g/l when grown in regular BG-11, Figure 2, and up to 0.74 g/l when grown in BG-11 with bicarbonate, Figure 3.
Although the JA06 strain already showed some promising numbers for acetate production it was believed that this number could have been increased further with the genetic modifications that were planned for the organism. None of the planned modifications were however successfully performed as there were major problems with the assembly and transformation of constructs during the project.
Growth trials of production organisms on acetate
A growth trial was performed to investigate the ability of the different organisms to naturally grow on acetate in BG-11 media. The organism that was most successfully grown under the desired conditions was Y. lipolytica, Figure 4. Some of the organisms, like B. subtilis, were not expected to grow on acetate as certain modifications are needed to make it compatible. But with all the issues that were encountered with the construct assemblies and transformations there was not time to get them working.
Y. lipolytica showed a fast stable growth at 5 g/l of acetate, but was able to grow in slightly lower concentrations as well. Considering that the current highest obtained concentration of acetate was 0.74 g/l a stable co-culture between the two organisms is not quite possible yet. With some of the modifications that were planned for the organisms it is possible that the acetate production would’ve been enough for a stable growth.
Co-culture growth trial
Promoter study
The results from the promoter study is presented in the page dedicated to describing the promoter study.
