RBS + cscB (codon optimized for cyanobacteria)
This part features the RBS + Spacer derived from Synechocystis sp. PCC 6803, and cscB (sucrose permease), optimized for Synechococcus elongatus PCC 7942.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 498
Illegal NgoMIV site found at 621
- 1000COMPATIBLE WITH RFC
In order to test CscB (sucrose permease), we added 150 mM of NaCl to 250 mL erlenmeyer flasks with 50 mL of BG-11 to induce sugar production. S. elongatus PCC 7942 naturally produces sucrose to balance out the osmotic pressure caused by the extracellular salt. Additionally, we added 2 g/L of pH 8.0 HEPES buffer to prevent acidification of the media (as CscB depends on a basic environment to function). We also induced with 1 mM of IPTG after the cells had reached the mid-log phase. The Sucrose/D-glucose Assay Kit was donated to us by Megazyme and was tested on the cyanobacteria after 1 week and then 2 weeks of IPTG induction. The cyanobacteria were spun down at 10,000 g for ~5-10 minutes until a pellet formed, and 200 µL of their supernatant was extracted for the assay.
With the assay, the sample was mixed with both acetate buffer (negative control) and fructosidase. After incubating at 50 ℃ for 20 minutes, we added the GOPOD dye, which turns a bright pink (510 nm) when exposed to glucose. After incubating for the required time, we ran triplicates in a spectrophotometer and calculated glucose values with the calculator on Megazyme’s website.
We tested all of our samples against glucose and starch controls (0.25g/L of flour), as well as uninduced samples of cyanobacteria (which lacked NaCl and/or IPTG).
After the insignificant results, we measured the pH because CscB will not export sucrose in acidic conditions. Before even testing for sucrose, we noticed that the wild type culture (without antibiotics) had gotten visibly contaminated by fungus [Figure 2]. As such, we suspect contamination to be the major cause of our lack of results.
Then, we started a new round of testing, with and without added antibiotics. We conducted the sucrose assay as before, except the cultures with antibiotics were supplied with 10 µL spectinomycin and 10 µL streptomycin. The cultures we measured sucrose for are:
- 1 = cscB #3 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 25 ℃
- 2 = cscB #2 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 25 ℃
- 3 = cscB #2 0 mM NaCl 1 mM IPTG 33 ℃
- 4 = cscB #2 150 mM NaCl 1 mM IPTG 33 ℃
- 5 = cscB #2 150 mM NaCl 33 ℃
- 6 = cscB #4 #1 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
- 7 = cscB #4 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
- 8 = cscB #2 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
- 9 = cscB #4 #2 150 mM NaCl 1 mM IPTG 33 ℃
Figure 3 shows the data for the sucrose d-glucose assay on the cyanobacteria after 1 week of IPTG induction while Figure 4 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 3 and 4. The induced samples showed no significant extracellular sucrose production compared to the starch controls.
After the insignificant results, we measured the pH [Table 1]. The pH was >7 for all cultures, so we suspected the lack of sucrose to be due to contamination or lack of selection pressure.
After addition of antibiotics to our culture, some values were positive for sucrose, but were likely due to leftover traces [Figure 5]. Sucrose 2 is likely statistical anomaly and none of the other results were statistically significant.
We repeated these experiments with 100mM of IPTG; however, we did not detect noticeable sucrose concentrations.