Week1(8/24/2016-8/30/2016)

Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)
The mutations of PETase gene had been contracted by over-lap PCR and Restriction Enzyme cutting sites(SacⅠ&EcoRⅠ) had been added to it’s ends (5’&3’) separately.

Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1(with AmpR) and plating on solid LB culture medium with Amp.

Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.

Plasmid isolation of pRset_CFP-1.

Primers were designed for the target (CFP gene) within the plasmid pRset_CFP-1.

PCR with Q5-Polymerase to amplify CFP gene out of plasmid pRset_CFP-1 and add Restriction Enzyme cutting sites(XbaⅠ&SacⅠ) to it’s ends(5’&3’) separately.
Bands as expected(about 760bp) in agarose gel , gel purification of the PCR products.

Week2(8/31/2016-9/6/2016)

XbaⅠ&SacⅠdouble restriction endonuclease digestion in CFP gene.

XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1
Bands as expected(760bp&3000bp) in agarose gel , gel purification of the digested products(3000bp).

Ligaion of digested pRset_CFP-1, digested CFP gene and digested PETase(M154L) gene in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-M154L.
Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1-M154L(with AmpR) and plating on solid LB culture medium with Amp.
Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of E.coli with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel.

Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.

Plasmid isolation of pRset_CFP-1-M154L.

Week2(8/31/2016-9/6/2016)

XbaⅠ&SacⅠdouble restriction endonuclease digestion in CFP gene.

XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1
Bands as expected(760bp&3000bp) in agarose gel , gel purification of the digested products(3000bp).

Ligaion of digested pRset_CFP-1, digested CFP gene and digested PETase(M154L) gene in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-M154L.
Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1-M154L(with AmpR) and plating on solid LB culture medium with Amp.
Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of E.coli with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel.

Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.

Plasmid isolation of pRset_CFP-1-M154L.

Week4(8/31/2016-9/6/2016)

XbaⅠ&SacⅠdouble restriction endonuclease digestion in CFP gene.

XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1
Bands as expected(760bp&3000bp) in agarose gel , gel purification of the digested products(3000bp).

Ligaion of digested pRset_CFP-1, digested CFP gene and digested PETase(M154L) gene in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-M154L.
Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1-M154L(with AmpR) and plating on solid LB culture medium with Amp.
Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of E.coli with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel.

Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.

Plasmid isolation of pRset_CFP-1-M154L.

Week5(8/31/2016-9/6/2016)

XbaⅠ&SacⅠdouble restriction endonuclease digestion in CFP gene.

XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1
Bands as expected(760bp&3000bp) in agarose gel , gel purification of the digested products(3000bp).

Ligaion of digested pRset_CFP-1, digested CFP gene and digested PETase(M154L) gene in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-M154L.
Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1-M154L(with AmpR) and plating on solid LB culture medium with Amp.
Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of E.coli with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel.

Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.

Plasmid isolation of pRset_CFP-1-M154L.

Week6(9/28/2016-10/2/2016)

Substrate:0.2mM pNPA water solution.
10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.
After static reaction at 39℃ for 8 hours, we detected the characteristic adsorption peak of the product ,pNP, which has no other characteristic adsorption peak except in 400nm.

Substrate:PET.
PET was been put into 20 times diluted Enzyme solution(unpurified).
After static reaction at 39℃ for 48 hours, we detected the characteristic adsorption peak of the product ,MHET, which has no other characteristic adsorption peak except in 260nm. Besides, we also deteected the system by Multispectral Scanning.

Team:Tianjin/Note/CFPS

Week1(8/24/2016-8/30/2016)