Team:RDFZ-CHINA/Result
Result
Demonstration:
In this section, we will mainly explain the planned flow of the experiments and our approach to validation of components. However, due to COVID 19 and laboratory constraints, we were forced to suspend our experiments several times, which made our experiments progress very slowly and eventually only stopped at the build stage. Therefore, we will also give the results of the experiments under the assumptions in the respective headings. Further information about testing every part is in engineering success (https://2021.igem.org/Team:RDFZ-CHINA/Engineering) Construction result a. DSF system construction (BBa_K3820020):
DSF-sensoring fragment synthesized on pUC57 was amplified by PCR restriction using DSF-F and DSF-R primers.
The PCR product of DSF-sensoring and the PCR product of pBBR-vec fragment were subjected to Gibson assembling.
This year 's process: suspended at Gibson assembling. b. pBBR plasmid cutting (BBa_K3820011):
On the pBBR plasmid we found suitable sticky sites with the help of snapgene and planned to unfold the plasmid into linearity by PCR to help us construct other components onto the pBBR plasmid.
Using pBBR-vec-R and pBBR-vec-F, the pBBR plasmids were unfolded into linear DNA sequences by PCR and used for insertion into the DSF-sensoring system.
Using pBBR-GdpX1-plasmid-F and R primers, the pBBR plasmid unfolded into a linear and used to insert GdpX1 sequence by PCR.
c. GdpX1 system construction (BBa_K3820021):
GdpX1 fragments synthesized on pUC57 were restricted amplification by PCR using GdpX1-F and R primers.
This year's process: suspended Gibson assembling.
Gibson assembling using PCR products and pBBR plasmid sheared PCR products. d. dCas12a system constructs (BBa_K3820024, BBa_K3820025, BBa_K3820026, BBa_K3820023, BBa_K3820022).
BBa_K3820024: HTH-type transcriptional regulator CymR, plasmid comes with, no need to construct.
BBa_K3820025: endolysin expression gene sequence. CymR fragment was replaced with endolysin using PCR and gibson ligation technique.
BBa_K3820026: tail protein expression gene sequence. Replacement of CymR fragments with major tail protein using PCR and gibson ligation techniques.
BBa_K3820023: sgRNA expression gene sequence. The two synthetic sgRNAs were inserted using PCR as well as gibson ligation technique.
BBa_K3820022: dcpf1 expression gene sequence. This plasmid is already completed, no need for construction.
e. Primer list:
In this section, we will mainly explain the planned flow of the experiments and our approach to validation of components. However, due to COVID 19 and laboratory constraints, we were forced to suspend our experiments several times, which made our experiments progress very slowly and eventually only stopped at the build stage. Therefore, we will also give the results of the experiments under the assumptions in the respective headings. Further information about testing every part is in engineering success (https://2021.igem.org/Team:RDFZ-CHINA/Engineering) Construction result a. DSF system construction (BBa_K3820020):
DSF-sensoring fragment synthesized on pUC57 was amplified by PCR restriction using DSF-F and DSF-R primers.
The PCR product of DSF-sensoring and the PCR product of pBBR-vec fragment were subjected to Gibson assembling.This year 's process: suspended at Gibson assembling. b. pBBR plasmid cutting (BBa_K3820011):
On the pBBR plasmid we found suitable sticky sites with the help of snapgene and planned to unfold the plasmid into linearity by PCR to help us construct other components onto the pBBR plasmid.
Using pBBR-vec-R and pBBR-vec-F, the pBBR plasmids were unfolded into linear DNA sequences by PCR and used for insertion into the DSF-sensoring system.
Using pBBR-GdpX1-plasmid-F and R primers, the pBBR plasmid unfolded into a linear and used to insert GdpX1 sequence by PCR.
c. GdpX1 system construction (BBa_K3820021):
GdpX1 fragments synthesized on pUC57 were restricted amplification by PCR using GdpX1-F and R primers.
This year's process: suspended Gibson assembling.
Gibson assembling using PCR products and pBBR plasmid sheared PCR products. d. dCas12a system constructs (BBa_K3820024, BBa_K3820025, BBa_K3820026, BBa_K3820023, BBa_K3820022).
BBa_K3820024: HTH-type transcriptional regulator CymR, plasmid comes with, no need to construct.
BBa_K3820025: endolysin expression gene sequence. CymR fragment was replaced with endolysin using PCR and gibson ligation technique.
BBa_K3820026: tail protein expression gene sequence. Replacement of CymR fragments with major tail protein using PCR and gibson ligation techniques.
BBa_K3820023: sgRNA expression gene sequence. The two synthetic sgRNAs were inserted using PCR as well as gibson ligation technique.
BBa_K3820022: dcpf1 expression gene sequence. This plasmid is already completed, no need for construction.
e. Primer list:
