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Team Cinnamaldehyde & Vanillin

Research Designs and Methods

The goal for cloning is to place each gene in the cinnamaldehyde pathways under a constitutive pTET and arabinose promoter pBAD for future characterization of each part. Our final construct should consist of a single pSB1C3 plasmid with all the genes inserted along with its ribosomal binding site and terminator:

Primers for the PAL/TAL, Ligase, and reductase genes are designed to yield PCR products containing all biobrick cut sites in the correct order: Eco R1, Xba 1, Spe1, and PstI. These PCR products could then be digested for ligation into available plasmids containing promoters (with or without rbs) and terminators.

June 28th

Transformation of 4-coumarate-CoA Ligase (4CL) and caffeic acid-O-methyl transferase (COMT)

Experimenter: Joe

Aim: Transform biobricks 4CL ( 2013 Distribution Kit, Plate 2, Well 17P) and COMT (2013 Distribution Kit, Plate 5, Well 19D) for miniprep and later amplification through PCR

Results: Transformation done according transformation procedures. Competent cells were plated on LB+ Amp plates and grown overnight at 37C. Only 4-CL grew overnight. No colonies were observed for COMT. Inoculated a colony of 4CL for miniprep

Rhodobacter sphaeroides Inoculation

Experimenter: Joe

Aim: A colony of Rhodobacter sphaeroides was inoculated in 5mL LB culture for DNA extraction

July 30th

Miniprep 4CL

Experimenter: Joe

Aim: Miniprep 5mL innoculation of 4CL with Qiagen Miniprep Kit

July 4th

PCR of 4-CL

Experimenter: Joe

Aim: PCR to amplify 4-CL from biobrick with biobrick VF2 and VR primers to confirm the size of 4-CL

Results: Multiple bands were seen due to possible mispriming of VF2 and and VR.

July 4th

PCR of 4-CL

Experimenter: Joe

Aim: PCR to amplify 4-CL from biobrick with biobrick VF2 and VR primers to confirm the size of 4-CL

Results: Multiple bands were seen due to possible mispriming of VF2 and and VR.

Retransformation COMT

Experimenter: Joe

Aim: PCR to amplify 4-CL from biobrick with biobrick VF2 and VR primers to confirm the size of 4-CL

Results: Multiple bands were seen due to possible mispriming of VF2 and and VR.

July 4th

PCR of 4-CL

Experimenter: Joe

Aim: PCR to amplify 4-CL from biobrick with biobrick VF2 and VR primers to confirm the size of 4-CL

Results: Multiple bands were seen due to possible mispriming of VF2 and and VR.

August 19th

Experimenter: Anna Müller

Aim: Check if Fisal’s ligations were successful: TAL in PSBIC3 and TAL with arabinose promoter with colony PCR

Results: 2 colonies were picked for each. No bands were seen on the gel. Ligations were unsuccessful.

August 21st

Experimenter: Anna Müller Aim: Ligate PAL and COMP to a constitutive promoter with RBS (.22A) Results: After transforming, 2 colonies were picked and PCR’d (21st Aug. and 26th Aug.). The gel confirmed the presence of both PAL (2100bp) and COMT (1001bp).