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Contents 1 Team Cinnamaldehyde & Vanillin 1.1 Research Designs and Methods 1.2 June 28th 1.2.1 Transformation of 4-coumarate-CoA Ligase (4CL) and caffeic acid-O-methyl transferase (COMT) 1.2.2 Rhodobacter sphaeroides Inoculation 1.3 July 30th 1.3.1 Miniprep 4CL 1.4 July 4th 1.4.1 PCR of 4-CL 1.4.2 Retransformation COMT 1.5 July 5th 1.5.1 Digestion of 4-CL 1.6 August 19th 1.7 August 21st 1.8 August 27th 1.9 August 29th 1.10 30th August 2013 1.11 August 31st 1.12 September 1st 1.13 September 2nd

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.

Retransformation COMT

Experimenter: Joe

Aim: Retransformation of COMT from biobrick registry and plated on LB agar + Amp

Results: Only one colony found on plate. This could have been the competent cells not being very competent.

July 5th

Digestion of 4-CL

Experimenter: Joe

Aim: Digest 4-CL to see if 4-CL is inserted into pSB1C3 plasmid with EcoRI and PstI

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).

August 27th

Experimenter: Anna Müller

Aim: Digest PAL (cPCR product) with EcorI and Spe I so it can be ligated to the terminator.

Results: No bands were seen on the gel run. It was concluded to go back to the plate and inoculate from the colonies

that showed to contain PAL. The DNA then got cleaned up.

Experimenter: Anna Müller

Aim: Clean up COMT DNA to get it ready for the digest with Ecorl and Spe I

Results: Clean up successful.

Experrimenter: Anna Müller

Aim: PCR COMT from cleaned up DNA for digest

Results:Bands were seen on a gel.

Experimenter: Anna Müller Aim: Digest COMT with EcorI and Spe I Results: Digest was successful when run on the gel.

August 29th

Experimenter: Anna Müller

Aim: Prove the presence of AtCCRI in colonies plated by Fisal

Results: Two colonies were picked one of them showed to contain the wanted gene (1039bp)

Experimenter: Anna Müller

Aim: Clean up PAL DNA (with constitutive promoter)

Results: Clean up successful

Experimenter: Anna Müller

Aim: Extract PAL, 4-CMH and 4-CL gene from BioBrick plasmid

Results: For PAL (2430bp) and 4-CMH (1558bp) bands were seen on a gel. The 4-CL PCR reaction was unsuccessful.

30th August 2013

Experimenter: Anna Müller

Aim: Ligation of PAL and COMT (cut by X bal and Pst I ) to arabinose promoter

Results: will follow

Experimenter: Anna Müller

Aim: Digest of AtCCRI and 4-CMH with X bal and Pst I to then ligate to constitutive promoter and arabinose promoter

Results: will follow

Experimenter: Anna Müller

Aim: Digest PAL with EcorI and Spe I to ligate it to a terminator

Results:Successful

Experimenter:Anna Müller

Aim: Digest AtCCRI and 4-CMH with Xbal and PstI to ligate to promoter.

Results:Digest succesful

Experimenter: Anna Müller

Aim: Digest PAL with EcorI and SpeI

Results:Digest failed multiple bands when run on gel. Possible star activity of the restriction enzymes.

Experimenter: Anna Müller

Aim: Ligation of PAL, COMT to arabinose promoter with rbs and AtCCRI, 4-CMH to arabinose and constitutive promoter:

Results:Ligations were successful -> proven on gel.

August 31st

Aim:Digest terminator with EcorI and Xbal, so it can be ligated to inserts cut with EcorI and SpeI

Results:Didn't work, all attempts to ligate failed. Should gel extract nexts time.

September 1st

Aim: cPCR the transformed and plated ligations done on August 31st.

Results:PAL w/arab, 4-CMH w/arab, 4-CMH w/const, COMT w/arab and AtCCR1 w/arab.

September 2nd