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 with 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.
|Knock-out argH + insert ackA||No|
|Knock-out glnA + insert AckA||No|
|Knock-out Acs + insert RFP||No|
|Knock-out Acs + insert pta||No|
|E. coli constructs||Assembled?|
|Overexpression of point mutated argA + GFP insertion||No|
|B. subtilis constructs||Assembled?|
|Y. lipolytica constructs||Assembled?|
|Overexpression of gln1 and GFP||No|
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
Two initial co-cultures were tested using the unmodified JA06 strain of Synechocystis and the WT of Y. lipolytica. In one of the co-cultures standard BG-11 media was used, Figure 5, and in the other BG-11 with 5 g/l of acetate was used, Figure 6. These co-cultures had their cell densities measured over time by using a microscope and a counting chamber.
As can be seen in Figure 5, the acetate production alone in JA06 is not enough to sustain a stable growth of Y. lipolytica, but can rather only keep a few cells alive. This was expected based on the growth trials of Y. lipolytica and JA06. It is however positive to see that the co-culture with added acetate seems to show that both organisms can be sustained in a single culture without causing any obvious problems for the other’s survival.
It is worth noting that the cell density differs by a lot from day to day. This can mostly be contributed to inexperience of the team members in using counting chambers and the fact that cell density differed much depending on which team member counted that day.
The results from the promoter study is presented in the page dedicated to describing the promoter study.
To further develop this project, a few things should be considered. Firstly, there have been reports of Synechococcus elongatus strains that accumulate sucrose to combat osmotic stress . This could be a better choice of product for the cyanobacteria in the co-culture. To support excretion of the sucrose, additional transporters could be added . Y. lipolytica has been shown to grow on sucrose after adding a gene for sucrose utilization .
Secondly, for Synechosystis it might be easier and quicker to knock down the expression of targeted genes than knocking them out, since the multiple copies of chromosomes in Synechosystis makes knocking genes out a labor intensive and slow process .
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