We did not manage to successfully grow our S. elongatus strain UTEX2973. Optimal growth requires BG-11 media, adequate light (400 μmol m-2 s-1) and CO2 and a temperature of 39-41°C. However, over a range of light (50-400 μmol m-2 s-1) and temperature conditions (30-41°C) and BG-11 concentrations no growth was exhibited in either liquid culture or on BG-11 plates. While we did not have the desired control over CO2 concentration we expected growth even at low CO2 concentrations. Ascorbic acid and sodium thiosulfate were trialled as a media supplement in an attempt to eliminate reactive oxygen species (Michael Burgis - personal communication) that can inhibit growth but no effect was observed from this. It is possible our strain was dead due to being held in customs for months in non-optimal conditions.
S. Elongatus Gene Constructs
We had successful transformation of our first S. elongatus construct containing genes 1-3 for the tropine biosynthesis pathway into our E. coli cloning strain and possible transformation of the second construct (containing genes 4-6). Gibson assembly of the two constructs were carried out and these constructs were then introduced into our electrocompetent E. coli cloning strain. These cells were then plated onto antibiotic plates and in each case multiple colonies exhibited antibiotic resistance. Thus indicating successful transformation of the plasmid. Three Colonies were picked for each plasmid and tested via PCR to determine whether our genes were correctly placed in the plasmid. Two PCRs were carried out for each of our constructs with primers that overlapped the region between our introduced gene sets and the backbone plasmids. The setup for the PCR gels is displayed in the table below and the gels are given in the two figures below.
PCR Gel Loading
|S. Elongatus Construct 1, gens 1-3 (figure 1):|
|Hyper ladder 1kb||PP1, Se 1-3 colony 1||PP1, Se 1-3 colony 3||PP1, Se 1-3 colony 4||PP1 control||PP2, Se 1-3 colony 1||PP2, Se 1-3 colony 3||PP2, Se 1-3 colony 4||PP2 control|
|S. Elongatus Construct 2, genes 4-6 (figure 2):|
|Hyper ladder 1kb||PP3, Se 4-6 colony 2||PP3, Se 4-6 colony 3||PP3, Se 4-6 colony 4||PP3 control||PP4, Se 4-6 colony 2||PP4, Se 4-6 colony 3||PP4, Se 4-6 colony 4||PP4 control|
E. coli Gene Constructs
We did not manage to successfully transform the two E. coli gene constructs into our E. coli cloning strain. Gibson assembly was carried out to form both of the plasmid constructs containing genes 1-3 and 4-6 respectively for the tropine biosynthesis pathway in E. coli. These were then transformed into our electrocompetent E. coli cloning strain however no colonies displayed antibiotic resistance, thus indicating no form of the plasmids (with or without our added gene sets) were successfully introduced to the cells.
Given that the S. Elongatus plasmids were successfully transformed into our E. Coli cloning strain suggests the difficulty with the E. Coli plasmids were not at the gibson assembly stage. Instead we suggest that since the E. coli plasmids contain E. coli promoters while the S. elongatus plasmids do not, that the metabolic burden on the cloning strain of both expressing the enzymes on the plasmid and replicating the plasmid led to non-viable cells. To combat this in future we could consider having inducible E. Coli promoters on these plasmids, then expression could be inhibited in the cloning strain reducing the metabolic burden of the plasmid and hopefully leading to viable cells for cloning.