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Results

In order to determine a growth curve for our strain of cyanobacteria, we measured the daily optical density (OD₇₅₀) of the cyanobacteria.

Figure 1 Growth curve of 1% UTEX cyanobacteria culture for two replicates (1m and 2m). Measured at an absorbance of 750 nm.

The results for the growth experiments at room temperature showed that the lag phase was around 103 hours [Figure 1]. After log-transforming the data and omitting lag, the doubling time for the room temperature cyanobacteria was found to be around 49 hours.

Sodium bicarbonate experiments

In order to determine the effect of both sodium bicarbonate and temperature, we conducted sodium bicarbonate experiments.

Figure 2 Sodium bicarbonate growth at 25℃

Figure 3 Sodium bicarbonate growth at 33℃

The sodium bicarbonate cultures showed the same growth curve as the experiments previously conducted. Compared to the 25℃, the 33℃ had a much faster growth rate. The doubling time for no bicarbonate at 33℃ at was 34 hours, which is already shorter than the 49 hours doubling time from the first growth experiments at 25℃. The doubling times for 5 mM bicarbonate at 33℃ is 31 hours, 10 mM at 33℃ is 30 hours, and 20 mM at 33℃ is 33 hours. The trend in doubling time indicated that increasing the concentrations of sodium bicarbonate up to 10 mM increased the growth rate, but after 20 mM it decreased the growth rate [Figures 2, 3]. We, therefore, concluded that 10 mM of sodium bicarbonate solution is the optimal concentration to add to our growth media.

Luciferase Assays

Constitutive Promoters: cpc and cpc-560 experiments

Figure 4 Daytime Standard Expression of luxAB for the constitutive promoters cpc and cpc-560.

Figure 5 Daytime and nighttime standard expression tests for the constitutive promoters cpc and cpc-560.

After removing outliers from the data set using the 1.5(IQR) rule and conducting unpaired T-tests assuming unequal variance, both cpc and cpc-560 showed significant differences for daytime expression compared to wild type [Figure 4]. Compared to cpc, cpc-560 had a significantly higher expression [Figure 4]. In comparison for day compared to night, cpc did not show any significant difference [Figure 5]. On the other hand, cpc-560 showed a significant difference between daytime and nighttime expression [Figure 5].

Regulated Promoters: idiA and psbA2 experiments

Figure 6 Daytime Standard Expression of luxAB for the constitutive promoters cpc and cpc-560, ferrous ion repressible idiA, and high light inducible psbA2 promoters

Figure 7 High light exposure Standard Expression tests for the constitutive promoters cpc and cpc-560, ferrous ion repressible idiA, and high light inducible psbA2 promoters

After removing outliers from the data set using the 1.5(IQR) rule and conducting unpaired T-tests assuming unequal variance, for daytime expression, compared to all other promoters and the wild type, cpc and cpc-560 had significant differences in expression [Figure 6]. For idiA and psbA2, they only had significant differences compared to cpc and cpc-560, and not to each other [Figure 6]. When exposed to high light (light >500 mM photons/m^2/s), idiA and psbA2 did not show any expression [Figure 7] Note: pre-light controls were not exposed to light.

Figure 8 Effect of Iron Chelator on expression for the constitutive promoters cpc and cpc-560, ferrous ion repressible idiA, and high light inducible psbA2 promoters

After removing outliers from the data set using the 1.5(IQR) rule and conducting unpaired T-tests assuming unequal variance, for expression after iron repression, idiA, psbA2, and cpc560 all showed a significant difference between before iron chelator reagent addition and after [Figure 8]. cpc’s insignificant difference could be attributed to the fact that there were only two cpc samples, as a result of insufficient cyanobacterial growth. The significant decrease in the expression of cpc560, however, as a result of adding the iron chelator, indicates that the cells may have died from prolonged deficiency of ferrous ions, therefore that one hour may have been too long [Figure 8].

Figure 9 Standard Expression tests under altered light conditions for the constitutive promoters cpc and cpc-560, ferrous ion repressible idiA, and high light inducible psbA2 promoters

When conducting our ferrous standard expression experiments, we had left the untreated cyanobacteria as well as the treated cyanobacteria in our biosafety cabinet, not expecting any major changes in expression due to the light exposure. However, under these light conditions, psbA2 was expressed significantly more than wild type [Figure 9]. We propose that this may be due to our incubators having excess light intensity or red light, which may have inhibited psbA2 expression.

Sucrose Assays

Figure 10 Results for the sucrose assay after 1 week of IPTG induction for cscB colonies.

Figure 11 Results for the sucrose assay after 2 weeks of IPTG induction for cscB colonies

Figure 10 shows the data for the Sucrose D-glucose assay on the cyanobacteria after 1 week of IPTG induction while Figure 11 shows the data for Sucrose D-glucose assay on the cyanobacteria after 2 weeks of IPTG induction. The controls for the experiment, glucose and starch, and uninduced samples of cyanobacteria (which lacked NaCl and/or IPTG) were not included in Figures 10 and 11. The induced samples showed no significant extracellular sucrose production compared to the starch controls.

After the insignificant results, we measured the pH. The pH was >7 for all cultures, so we suspected the lack of sucrose to be due to contamination or lack of selection pressure (Figure 1).

Table 1 pH testing of cscB cultures

After addition of antibiotics to our culture, some values were positive for sucrose, but were likely due to leftover traces. Sucrose 2 is likely statistical anomaly and none of the other results were statistically significant [Figure 12]. We repeated the experiment with 100 mM of IPTG; however, we did not detect noticeable levels of sucrose

Figure 12 Results from sucrose assay after addition of antibiotics

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