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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 and grown at 30C

Results: Culture grew to high density after a couple days

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 present in the pSB1C3 plasmid with EcoRI and PstI

Results: pSB1C3 plasmid is about 2070bp while 4CL gene is about 1656 bp. Digest was successful. Two bands were observed: one at 2kb and one at 1.7kb

Re-run PCR of 4CL

Experimenter: Joe

Aim: Re-run PCR of 4CL with fresh PCR reagents

Results: Mispriming seen but the brighter band is about 2kb, which is the size the PCR product of 4CL with the extensions from pSB1C3 (~1951bp)

July 6th

Genomic DNA extraction of R.sphaeroides grown on June 28th

Experimenter: Joe

Aim: Bacterial DNA extraction using QuickExtract Bacterial DNA Extraction Kit

Results: DNA extraction were done following the above kit and diluted in TE buffer

July 8th and 9th

Inoculation of COMT and miniprep

Experimenter: Joe

Aim: Inoculated one colony of COMT in 5mL LB + Amp and grew overnight for Miniprep.

Results: Miniprepped the 5mL innoculation of COMT with Qiagen Miniprep Kit and eluted with 30uL of dH2O

Received PAL, 4-CMH from iGEM HQ

Experimenter: Joe

Aim: Plated cells received form iGEM HQ. These include BBa_K801091 (Phenylalanine ammonia lyase-PAL) and BBa_K238007 (4-coumarate 3-hydroxylase) which were plated on Chlor LB plates and Amp LB plates respectively.

Results: Cells grew on their respective plates and inoculated with 5mL of the LB on their respectively antibiotic. Miniprepped the 5mL inoculation of COMT with Qiagen Miniprep Kit and eluted with 30uL of dH2O

July 8th and 9th

Genomic DNA extraction of R.sphaeroides grown on June 28th

Experimenter: Joe

Aim: Bacterial DNA extraction using QuickExtract Bacterial DNA Extraction Kit

Results: DNA extraction were done following the above kit and diluted in TE buffer

July 8th and 9th

Genomic DNA extraction of R.sphaeroides grown on June 28th

Experimenter: Joe

Aim: Bacterial DNA extraction using QuickExtract Bacterial DNA Extraction Kit

Results: DNA extraction were done following the above kit and diluted in TE buffer

July 8th and 9th

Genomic DNA extraction of R.sphaeroides grown on June 28th

Experimenter: Joe

Aim: Bacterial DNA extraction using QuickExtract Bacterial DNA Extraction Kit

Results: DNA extraction were done following the above kit and diluted in TE buffer

July 8th and 9th

Genomic DNA extraction of R.sphaeroides grown on June 28th

Experimenter: Joe

Aim: Bacterial DNA extraction using QuickExtract Bacterial DNA Extraction Kit

Results: DNA extraction were done following the above kit and diluted in TE buffer

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

Experimenter:Anna Mueller

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

Experimenter:Anna Mueller

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 worked out.

September 2nd

Experimenter:Anna Müller

Aim:cPCR run on a gel.

Results:Ladder was unclear gel was run again, the gel confirmed PAL w/arab, 4-CMH w/arab, 4-CMH w/const, COMT w/arab and AtCCR1 w/arab worked

September 3rd

Experimenter:Anna Müller

@@ Line 287: / Line 287: @@
-'''Aim:'''Digest terminator with EcorI and Xbal, so it can be ligated to inserts cut with EcorI and SpeI.
+'''Experimenter:'''Anna Mueller
+'''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'''==
+'''Experimenter:'''Anna Mueller
 '''Aim:''' cPCR the transformed and plated ligations done on August 31st.
@@ Line 301: / Line 306: @@
 =='''September 2nd'''==
+'''Experimenter:'''Anna Müller
 '''Aim:'''cPCR run on a gel.
@@ Line 308: / Line 315: @@
 =='''September 3rd'''==
+'''Experimenter:'''Anna Müller

Team:British Columbia/Notebook/Flavours

From 2013.igem.org

Revision as of 03:21, 27 September 2013

Contents

Team Cinnamaldehyde & Vanillin

Research Designs and Methods

June 28th

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

Rhodobacter sphaeroides Inoculation

July 30th

Miniprep 4CL

July 4th

PCR of 4-CL

Retransformation COMT

July 5th

Digestion of 4-CL

Re-run PCR of 4CL

July 6th

Genomic DNA extraction of R.sphaeroides grown on June 28th

July 8th and 9th

Inoculation of COMT and miniprep

Received PAL, 4-CMH from iGEM HQ

July 8th and 9th

Genomic DNA extraction of R.sphaeroides grown on June 28th

July 8th and 9th

Genomic DNA extraction of R.sphaeroides grown on June 28th

July 8th and 9th

Genomic DNA extraction of R.sphaeroides grown on June 28th

July 8th and 9th

Genomic DNA extraction of R.sphaeroides grown on June 28th

August 19th

August 21st

August 27th

August 29th

30th August 2013

August 31st

September 1st

September 2nd

September 3rd