Part:BBa_K4595024
Prbcl-dsup-Tpsbc-Prbcl-gshA-gshB-Tpsbc
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 145
Illegal EcoRI site found at 2968 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 145
Illegal EcoRI site found at 2968
Illegal NotI site found at 4051
Illegal NotI site found at 4057
Illegal NotI site found at 4288
Illegal NotI site found at 4576 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 145
Illegal EcoRI site found at 2968
Illegal BamHI site found at 1210
Illegal BamHI site found at 1683
Illegal BamHI site found at 2187 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 145
Illegal EcoRI site found at 2968 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 145
Illegal EcoRI site found at 2968
Illegal NgoMIV site found at 2164
Illegal NgoMIV site found at 3450
Illegal NgoMIV site found at 3714
Illegal NgoMIV site found at 3732
Illegal NgoMIV site found at 3942
Illegal NgoMIV site found at 4299
Illegal NgoMIV site found at 4317 - 1000COMPATIBLE WITH RFC[1000]
Description
It is a complex component consisting of the promoter Prbcl, the target genes gshA, gshB, dsup, and the terminator Tpsbc. gshA and gshB are responsible for the high expression of Gsh synthesis, thus making intracellular GSH redundant in the engineered bacteria. dsup is responsible for promoting the synthesis of Damage suppressor protein in cells, improving the DNA strength of engineered bacteria, and enhancing their reproductive activity to resist the extreme environment that may be brought by the increasing climate crisis.
Prbcl
Pcpc560 is a super-strong promoter containing two predictive promoters from the cpcB
gene and 14 predictive transcription factor binding sites (TFBSs). This efficient promoter
enables heterologous proteins to be expressed robustly in Synechocystis
PCC6803.
dsup
The dsup gene sequence encodes for the Damage suppressor protein in Ramazzottius varieornatus, an organism known for its ability to thrive in extreme environments. Due to its resilience, various proteins found in Ramazzottius varieornatus are of interest for research on biological stress resistance. Notably, dsup protein has shown promising results in aiding cellular engineering by selectively binding to specific regions of DNA. By inhibiting X-ray induced DSBS and SSBS, Dsup can support the reproductive activity of engineered cells. Our goal is to apply this method in enhancing the resilience and proliferation of our engineered bacteria in extreme environments. To achieve this, we introduced the dsup gene into Synechocystis PCC6803 and performed codon optimization.
gshA and gshB
Glutathione (GSH) is an important antioxidant containing sulfur compounds. It is a tripeptide composed of three amino acids (cysteine, glycine, and glutamic acid) and non-protein thiols. GSH is usually in a reduced state and plays a critical role in detoxifying reactive oxygen species (ROS) and free radicals that cause oxidative stress. ROS can change the spatial structure of proteins by obtaining electrons, leading to the degeneration of many protein-dependent chemical substances and even DNA, further affecting the normal life activities of the cell. However, the reduced form of GSH can protect the chemical structure of proteins by providing extra electrons for ROS and free radicals. GSH peroxidase catalyzes this process. According to the reference, we founded that Arthrospira sp. PCC 8005 activates the cell's Gsh synthesis pathway to resist radiation hazards such as oxde-induced protein denaturation and DNA damage rather than inducing classical antioxidant or DNA repair systems like superoxide dismutase (SOD) enzymes and RecA proteins. To enhance our engineered bacteria's resistance to extreme environments, we overexpressed GSH synthesis in advance in Synechocystis PCC6803.This measure was aimed at equipping the bacterium with the capacity to withstand potential threats arising from the escalating climate crisis. By enhancing its adaptation to extreme environments, this modified strain may not only survive but also potentially mitigate any adverse effects. Moreover, it has the potential to offer insights into the reclamation of dangerous land areas and the exploration of space.There is a natural Gsh synthesis pathway in Synechocystis PCC6803 mediated by two ATPdependent reactions which catalyzed by γ-glutamylcysteine synthelase (γ-GCs) and glutathione synthase (GS). γ-Gcs and GS are encoded by two different genes, the gshA and gshB genes, respectively. The gshA gene sequence encodes γ-glutamylcysteine synthelase (gamma-GCs), which binds glutamate and cysteine to form γ-glutamylcysteine metabolites. gshB is the gene sequence encoding glutamine synthetase (GS), which generates glutathione by adding glycine to γ-glutamylcysteine metabolites (Fahey, 2013).
Molecular cloning
In order to construct the desired plasmids, we employed the E.coli TOP10 amplification
method. Firstly, we performed PCR amplification using specific primers for each plasmid,
which results in the generation of linearized fragments harboring the target sequences in
a high copy number. These fragments were then connected into complete plasmids using
restriction enzyme digestion and enzyme ligation procedures. After transfer to E.coli TOP10,
colony PCR was used to confirm successful construction of the plasmid. Subsequently, the
plasmids were further amplified to obtain sufficient quantities for further experiments.
Finally, the complete plasmids were introduced into Synechocystis
PCC6803 cells and their
successful integration was verified through colony PCR analysis.
The band of Prbcl-dsup-Tpsbc-Prbcl-gshA-gshB-Tpsbc from colony PCR is about 4700bp, identical to the theoretical length of 4746bp estimated by the designed primer location which could demonstrate that this target plasmid had successfully transformed into E.coli TOP 10.
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