•
The plasmid introduced into the cyanobacteria was obtained from the ShanghaiTech_China 2022
team. They
constructed a plasmid containing the Sucrose permease (cscB) gene, which can integrate into the
cyanobacterial genome. We expressed this plasmid in S. elongatus. For more information, please
check the
ShanghaiTech_China 2022 website at Engineering Success |
ShanghaiTech_China -
iGEM 2022
Cycle1: Culturing
Design
•
We decided to cultivate the cyanobacteria using BG11 liquid medium, inoculating the strains at a
ratio of
100:1. The cultures were maintained at room temperature under natural light conditions. The
optical density
at 750 nm (OD750) was measured daily using a spectrophotometer to plot the growth curve.
Build
Room temperature under natural light
Test
the cyanobacteria entered the logarithmic phase on day 15 and reached the stationary phase on
day 27.
Learn
25°C constant illumination incubator (6000 lux) led to a faster growth rate
The constant illumination incubator (6000 lux) led to a faster growth rate
Cycle2: Stain Transformation
Design
•We reviewed literature to obtain the cscB gene sequence
for sucrose secretion and had a plasmid with regulatory elements synthesized.
Build
•We cultured 1.5 ml of cyanobacteria (OD750 = 0.5),
centrifuged, resuspended in BG11 medium, and added plasmid (≥1 ng/μl). The mixture was incubated
at 30°C for 24 hours.
Test
•After 24 hours, we spread the culture on BG11 agar plates
with antibiotics and incubated until single colonies appeared, confirming successful
transformation.
Learn
•We identified transformed colonies using antibiotic
selection, measured sucrose production, and adjusted conditions to optimize cscB expression,
documenting the process throughout.
Cycle3: Expression Validation
Design
•We selected engineered PCC7942 strains for cscB gene
expression experiments, planned their cultivation in BG11 medium with ampicillin, and prepared
the necessary equipment and reagents, including a sucrose assay kit.
Build
•Single colonies from antibiotic-resistant plates were
inoculated into BG11 medium with ampicillin and cultured for seven days until OD685 reached 0.5
to ensure sufficient growth and gene expression.
Test
•50 ml cultures of wild-type and engineered PCC7942 were
centrifuged, resuspended in BG11 medium, and sucrose levels were measured every 24 hours for 72
hours using a sucrose assay kit. Intracellular sucrose was also measured with additional
freeze-thawing, ultrasonication, and extraction steps.
Learn
•Data was analyzed to evaluate cscB gene expression and
sucrose production. Based on results, adjustments to the expression system or conditions were
made to improve future experiments.
References
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