Part:BBa_K656011
cscB Sucrose Symporter
cscB sucrose transporter from e.coli W
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 310
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 310
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 310
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 310
Illegal NgoMIV site found at 487 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 531
Characterization of cscB sucrose transporters in PCC7942
Measurement of growth and sucrose export in cscB-transformed PCC7942 along with assesment of transformation stability by omitment of antibiotic pressure - by Team Cosmocrops 2016, University of Copenhagen.
Background: Sucrose is a disaccharide composed of glucose and fructose that are linked together with alpha- (1,2) glycosidic linker. In photosynthetic organisms like cyanobacteria and plants, sucrose is naturally produced and stored by means of photosynthesis.
Characterization: The iGEM team from Brown-Stanford 2011 utilized a cscB coding gene inspired from the research of Daniel Ducat and Pamela Silver at Harvard Medical School. However, they were not able to perform sucrose measurements when their documentation ended, as described in their wiki-pages (<a> href="http://2011.igem.org/Team:Brown-Stanford/PowerCell/NutrientSecretion"</a>). We would therefore like to add sucrose measurements to their BioBrick pages. With the permission from the original authors, we borrowed a cscB-transformed PCC7942 S. elongatus and characterized growth and sucrose accumulation in the extracellular medium during 7 days of induction, at different osmotic pressures.
Experiment:
The S. elongatus PCC7942 was inoculated in 100 mL BG-11 media in a conical flask and incubated at 30C under 200 rpm shaking and light bulb. After 3 days of growth, 1,5 mM of IPTG was added to induce the expression of sucrose export cscB gene for sucrose transport into the media.
To validate the amount of sucrose secretion in different conditions, 17 different samples of induced S. elongatus PCC7942 were prepared with different osmolarity (0mM, 50mM, 100mM, 150mM, 200mM, 300mM).The amount of sucrose accumulated was validated with and without selection pressure. Optical density was measured at 730nm wavelength to see a correlation between selected osmolarity and growth.
The amount of sucrose secreted in the media was determined using Dionex- High-performance anion exchange chromatography - pulsed ampherometric detection (HPAEC-PAD) by using using CarboPac SA 10 (Thermo Scientific) column for fast separation of sugars. The running program was consisted by a constant flow rate of 0.35 ml/min and injection volume of 20 µL. Before injection of each sample, the column was washed with 0.5 M NaOH for 5 min, then equilibrated with water 10 min. The elution program consisted of an isocratic elution with water from 0 to 10 min, followed by a linear gradient up to 0.5 M NaOH from 10 to 15 min. Monosaccharides were detected using a pulsed amperometric detector (gold electrode) set on waveform A, according to manufacturer’s instructions, with a post-column addition of 1 M NaOH for detection. Monosaccharide and disaccharide standards were from Sigma and included D-Glc, D-Fru, and sucrose. For verification of the response factors, a standard calibration was performed before analysis of each batch of samples for measurement of sucrose content.
Results: Although some samples were lost post 78 hours, sucrose was observed to rise steadily in the samples above 100 mM NaCl (Fig. 1) , which is also expected from the literature (Ducat et al 2012). However, a sudden and consistent drop in sucrose concentration was observed for the S5 series with 200 mM NaCl. We hypothesized that it was either a technical artifact or due to loss of live bacteria. Measuring OD confirmed that cell death could potentially explain the drop, since the OD for 200 mM series dropped throughout the experiment (Fig. 2). Such drop is likely due to rerouting of the carbon flux to the extracellular, however, we were only able to confirm that the drop in OD was dependent on the osmotic pressures, and that 50-150 mM were able to keep the cells alive (at least in figure 2, however, figure 4 tells a different story. They are likely both true and within a certain variance, we couldn’t measure). We were also interested in measuring OD and sucrose accumulation when CAM was omitted to test the stability of the cscB transformation (Fig. 3 and 4). However, due to sample loss it was difficult to compare them after seven days. In conclusion, cscB transformed PCC7942 was able to accumulate up to 1.4 mM in the extracellular medium, and omitting antibiotic did not seem to lower the overall sucrose exportation during seven days of experimentation.
Figure 1: Sucrose accumulation with CAM: <i>S. elongatus was induced at various concentrations of NaCl and 0.1 mM IPTG. Chloramphenicol(CAM) is an antibiotic, and was present at 10 ug/ml. Measurements span over 7 days with induction at time 0. </i>
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</i>Figure 2: OD measurements without CAM. S. elongatus was induced at various concentrations of NaCl and 0.1 mM IPTG. CAM was present at 10 ug/ml. Measurements span over 7 days with induction at time 0 hrs. </i>
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Figure 3: Sucrose accumulation without CAM. S. elongatus was induced at various concentrations of NaCl and 0.1 mM IPTG. CAM was omitted to test the stability of our setup without antibiotics. Measurements span over 7 days with induction at time 0 hrs.
Figure 4: OD measurements without CAM: S. elongatus was induced at various concentrations of NaCl and 0.1 mM IPTG. CAM was omitted to test the stability of our setup without antibiotics. Measurements span over 7 days with induction at time 0 hrs.
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