Team:Victoria Wellington/Challenges

As has been a theme around the world, our lab work got interrupted in the starting phase due to the beginning of a Covid-19 Delta variant outbreak in New Zealand. Our plans for making good progress on the laboratory work were halted days before we were to begin. We were in a lockdown setting for nearly a month - beginning in late August. This resulted in a reduction in our possible work time by one half. Once restrictions relaxed after the 16th of September we were able to pick up from where we left off. But in particular the attempts to grow Synechococcus elongatus were significantly interrupted. Remaining restrictions on how many people were able to be working within the lab, again, hindered our progress.
Growth of Synechococcus elongatus:
The ability to effectively and consistently grow S. elongatus is a vital component of our project. Unfortunately this is something that we were not able to achieve. Given our attempts with various conditions, it was made evident that our culture of UTEX2973 was dead. There were multiple factors that may have contributed to this. The first was difficulties in getting the sample through border customs in a timely manner. It was held at the border for almost a month, contained wiithin an airtight vessel and without light, the time in which the sample was held for processing may have been enough to cause the strain to die. Assuming that this was not the case, a second factor being the storage method of the sample once in the lab may have been the cause. Even if the strain was to be alive, we are still awaiting approval from the Environmental Protection Authority (EPA) to allow for genetic manipulation of UTEX2973 and given this, would be unable to proceed any further than the growing of the strain. A confounding factor would have been supply chain issues (exacerbated by regional lockdowns) with acquiring a CO2 source. Going forward, we must prioritise streamlining the delivery of our S. elongatus strain and to be stringent with storage protocols.
Gibson Assembly:
The Gibson assembly of our gene fragments was not very successful. Our attempts at this were able to produce restriction digests of the expected sizes for the S. elongatus plasmids, but not for the Escherichia coli plasmids. Having a large number of fragments reduces the efficiency of Gibson assembly due to the probability of restriction induced overhang binding. In addition, there is likely latent expression of the plant genes in the E. coli cloning host EC100, affecting their fitness as they produce high copy numbers of plasmid. To continue, we must be able to devise a way in which we can assemble the fragments for the E. coli plasmids. Reducing the number of fragments per assembly, using an E. coli host strain that can tolerate expression of these plasmids, or using inducible promoters might increase cloning efficiency. A possibility is that our desired construct of three fragments per plasmid is too large. Having a large number of fragments reduces the efficiency of a Gibson assembly due to the probability of restriction induced overhangs binding. Reducing the number of fragments per assembly will increase the number of amplification and transformation steps which may accumulate errors and thus reduce our ability to successfully arrive at the triparental mating. By adjusting the concentrations used, time, and perhaps the design of the overhangs, this issue may be resolved.