Team:Peking/Notebook
Notebook
Protocol
- PCR Protocol
- Molecular Cloning Protocol
- DNA Extraction&Purification Protocol
- E. coli Tranformation Protocol
- Microfluidic_Protocol
- Bacteria_Cell_Lysis_Protocol
Before July
4.10~5.25
Designing our project through brainstorm and literature research about each idea proposed. Three alternative projects were selected and we decided to do demonstration of each one in smaller groups. Prepared experiment equipment and reagents.
5.26~7.12
Demonstration of the three alternative projects. Learning and testing molecular cloning methods including PCR, Gibson Assembly, Golden Gate Cloning, gel purification, plasmid extraction etc. . Designing of sgRNAs that target different sites. Molecular cloning of pSB4A5-pBAD-dCas9-J23119-sgRNA_library. Preliminary experiment of growth curve measuring according to OD600. Preliminary experiment of serial dilution. Preliminarily measuring the response curve of pBAD.
July
7.13~7.18
Project confirmed. Molecular cloning of CRISPRi system. Molecular cloning to replace pBAD with pTAC. Research about quorum sensing system and target other than bacterial genome, such as mitochondrial DNA and plasmid.
7.19~7.26
Molecular cloning of pSB4A5-pBAD-dCas9-T7-sgRNA. The plan of quorum sensing system confirmed. Measuring the response curve of pBAD. Using CRISPRi system to test our dCas9 and sgRNA part.
7.27~8.1
Molecular cloning of pSB3C5-pBAD-dCas9-J23119-sgRNA_library serial and pBAD-dCas9-T7-sgRNA-J23119-mRFP. Molecular cloning of quorum sensing parts F2620 and K1499500. Growth curve measuring according to OD600 and serial dilution test of partial pSB3C5/pSB4A5-pBAD-dCas9-J23118-sgRNA_library.
August
8.2~8.8
Molecular cloning related to plasmid copy number control. Molecular cloning of RPG-pBAD-B0034-dCas9-J23119-sgRNA_library serial. Modification and optimization of some previous plasmid (terminator optimizing etc.). Characterization of quorum sensing part in plate reader.
8.9~8.15
OD600 measuring of RPG-pBAD-B0034-dCas9-J23119-sgRNA_library serial. Preliminary experiment of qPCR to measure plasmid copy number. Further characterization of quorum sensing part.
8.16~8.25
Further literature research about bacterial DNA replication and growth. Modification and optimization of some previous plasmid. Characterization of quorum sensing part on agar plate. Molecular cloning of quorum-sensing induced dCas9 system. Preliminary experiment of microfluidic system.
8.26~8.31
Modification and optimization of some previous plasmid. Characterization of partial sgRNA of our system and quorum sensing system in microfluidic chips.
September
9.1~9.7
Trial of measuring the OriC-Ter ratio through qPCR. Molecular cloning to add ssrA tag behind dCas9 to some plasmids. Characterization of partial sgRNA of our system and quorum sensing system in microfluidic chips.
9.8~9.14
Measuring bacterial growth rate and observing morphology through blood counting chamber. Observing morphology reverse of bacteria carrying RPG-pBAD-dCas9-J23119-sgRNA_library. Partially characterization of our CRISPRri system and quorum sensing system in microfluidic chips. Molecular cloning to add ssrA tag behind dCas9 to some plasmids.
9.15~9.21
Molecular cloning to add ssrA tag behind dCas9 to some plasmids. Characterization of ssrA effect in plate reader. Measuring plasmid copy number under our system’s control by qPCR. Characterization of our CRISPRri system and quorum sensing system in microfluidic chips under more detailed conditions.
9.22~9.28
Further characterization of our CRISPRri system and quorum sensing system in microfluidic chips under more detailed conditions. Multiple-reverse test of our control system. Measuring the GFP yield per unit biomass of bacteria with our control system and a GFP gene.
October
9.29~10.5
Molecular cloning of pSB1C3-pTAC-FMO. Molecular cloning to add an mRFP gene after the plasmid copy number controlling system. Trial of distinguishing cell morphology using FCM. Trial of observing the DNA distribution of bacteria under the control of our system using confocal laser scanning microscope. Characterization of our CRISPRri system and quorum sensing system in microfluidic chips.
10.6-10.13
Observing morphology multiple reversion of bacteria under the control of our system with ssrA tag. Characterization of our CRISPRri system and quorum sensing system in microfluidic chips.
10.13-10.20
Demonstration of measuring the field of indigo and PHA in bacteria under the control of our system. Demonstration of the behavior of E. coli strain Nissle under he control of our system.