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Team:British Columbia/Notebook/Flavours
From 2013.igem.org
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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
June 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: PCR products were ran on a 0.8% agarose gel. 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: PCR products were ran on 0.8% agarose gel. 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 was 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 Phenylalanine ammonia lyase (PAL), 4-coumarate 3-hydroxylase (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; 4CM-H) 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 15th
PCR pTet (BBa_J23118) Constitutive promoter
Experimenter: Joe
Aim: PCR pTet constitutive promoter for ligation into the pSB1C3 with rbs
Results: PCR products were ran on 0.8% agarose gel. A band was seen at 1kb because RFP is attached the constitutive promoter
Transformation of Constitutive GFP
Experimenter: Joe
Aim: Transform with 2uL of Constitutive GFP (BBa_K608008, Plate 1 Well 5I and BBa_I13522, Plate 3 Well 5J) and plate on LB agar + Chlor
Results: GFP expressing colonies were observed on both plates and inoculated in 5mL LB+Chlor for an overnight culture. Overnight cultures were then miniprepped using the Qiaprep Spin miniprep kit.
Digestion of 4-CMH and PAL from pSB1C3
Experimenter: Joe
Aim: Digest 4-CMH and PAL biobrick with EcoRI and PstI to check for the correct band size
Results: Digested products were ran on a 0.8% agarose gel. Bands were seen at 1600bp for 4-CMH and 2100bp for PAL
Ligation of pTET constitutive promoter to pSB1C3 plasmid with rbs
Experimenter: Joe
Aim: Ligate pTET constitutive promoter to rbs in pSB1C3
Results: Transformation halted because the 1kb band seen was a constitutively expressed RFP. pTET constitutive promoter needs to be digested with SpeI and PstI and ligated with the insert (rbs)
July 16th
Extraction of PAL and COMT from Plasmid registry
Experimenter: Fisal
Aim: Frances and I used the PCR protocol to extract PAL and COMT form the PSB1C3 vector which was miniprepped by Joe. An annealing temperature of 60 degrees and an extension time of 45 seconds seemed appropriate. I was in the lab with Frances who has no wet Lab experience so this was also a teaching/learning opportunity.
Results: Nonspecific and weak bands were observed in a 1% agarose gel for the 4CMH and the COMT in accordance with our PCR protocol.
Extract TAL from Rhodobacter sphaeroides Using our PCR protocol
Experimenter: Fisal
Aim: PCR out the TAL gene, an annealing temperature of 67 degrees and an extension time of 2 min was chosen.
Results: PCR for TAL failed; The wrong annealing temperature was used.
July 17th
Digestion of pSB1C3 Constitutive GFP (5I)
Experimenter: Joe
Aim: Digest pSB1C3 GFP with EcoRI and SpeI, EcoRI and PstI, XbaI and SpeI, XbaI and PstI which will be the vector for future ligations into pSB1C3
July 19th
PCR rbs(BBa_B0034; Plate 1 Well 2M)
Experimenter: Joe
Aim: PCR rbs (BBa_B0034) from biobrick with VF2 and VR primers
Results:PCR products were ran on a 0.8% agarose gel. A band at 300bp was observed, which included extensions of pSB1C3
PCR Streptomyces coliecolor Cinnamate: Coenzyme A Ligase (ScCCL)
Experimenter: Joe
Aim: PCR ScCCL gene from genomic DNA of Streptomyces coliecolor with ScCCL forward and reverse primers
Results:PCR failed. It is uncertain whether there is genomic DNA of Streptomyces coliecolorprovided by Dr. Thompson
PCR Rhodobacter sphaeroides Tyrosine Ammonia Lyase (TAL)
Experimenter: Joe
Aim: PCR TAL gene from genomic DNA of Rhodobacter sphaeroides with RsTAL forward and reverse primers
Results: PCR successful. PCR products were ran on 0.8% agarose gel. A band at between 1.65kb and 2.0kb was observed. RsTAL is about 1.7kb
Re-attempt the TAL PCR
Experimenter: Fisal
Aim: Change the annealing temperature to 60degrees, run several PCR reactions with varying levels of DMSO because secondary structure of genomic DNA suspected to cause problems. Different MgCl2 levels were used as well to optimize PCR.
Results: The PCR product with 6% DMSO and 0.75uL MgCl2 in a 50uL reaction had the best results.
July 22nd
Purify and digest TAL PCR product
Experimenter: Fisal
Aim: Use the Quiagen PCR clean up kit to extract DNA., then cut TAL with EcoR1 and Pst 1. Cut the miniprepped PSB1C3 vector with EcoR1 and PST1, then and Ligate to PSB1C3 to the TAL digest after using a kit to clean up both digested products.
Results: TAL purification failed when confirmed with the nanodrop spectrophotometer. Discussion with other lab members led to the conclusion that the Quiagen kit we were using was old and the pH in the buffers was off.
July 23rd
Re-Attempt PCR COMT and PAL PCR
Experimenter: Fisal
Aim: Re-attempt COMT and PAL PCR because the tube from July 16th was lost.
Results: Successful PCR was confirmed though a 1% agarose gel and bands at 1.3kbp for COMT and 2.3 kbp
Re-attempt the TAL PCR
Experimenter: Fisal
Aim: Extract TAL from Rhodobacter sphaeroides using the same protocol that yielded success on July19th
Results: PCR was successful with expected bands at 1.5kbp
Digest TAL, COMT, and PAL PCR products
Experimenter: Fisal
Aim: Clean PCR products using the Quiagen PCR clean up kit and protocol. Cut clean DNA with PST1 and Xba1 then purify digests.
Results: Digests were run in accordance with our standard protocols, DNA was eluted in TE buffer from the Qiagen kit.
Digestion of the pTET Constitutive promoter from pSB1C3 and rbs from PCR
Experimenter: Joe
Aim: Digest pTET constitutive promoter (from pSB1C3) with XbaI and PstI and rbs (from PCR) with XbaI and PstI for ligation
Ligation of rbs from PCR into pTET constitutive promoter
Experimenter: Joe
Aim: Ligate a construct with pTET constitutive promoter and a rbs
July 24th
Transformation of Constitutive and rbs ligation
Experimenter: Joe
Aim: Transform Constitutive promoter and rbs ligation into competent cells and plate onto LB agar + Chlor
Results: Colonies were observed after 18 hours of growth at 37C.
PCR feruoyl-CoA synthetase (Fcs) gene from Pseudomonas putida genomic DNA
Experimenter: Joe
Aim: PCR Fcs gene from Pseudomonas putida
Results: PCR products were ran on 0.8% agarose gel. Multiple non-specific bands were observed.
July 29th
Run PCR of TAL, COMT and PAL as a backup
Experimenter: Fisal
Aim: Run TAL, COMT and PAL PCR reactions as described in entries dated july16th -22nd
Results: TAL PCR failed, the wrong primers were used. COMT and PAL succeeded with expected bands of 2.3 kbp and 1.3kbp for COMT
Ligate the Digested TAL construct to PSB1C3from July 23rd, transform ligation into E.coli
Experimenter: Fisal
Aim: Using our standard Ligation protocol ligate the TAL digest into PSB1C3 which was cut by Joe with PST1 and XBa1; T4 ligase was used. Once the ligation is complete transform the product into E.coli chemically competent cells, plate, and grow overnight at 37 degrees.
Results: A 10uL ligation reaction was carried out and incubated at room temperature for 4 hours, the product was then transformed into chemically competent E.coli, plated on LB Chlor plates and incubated at 37 degrees overnight as per our transformation protocol
Digest PAL and COMT with Xba1 and Pst1
Experimenter: Fisal
Aim: Clean PAL and COMT PCR products using the Qiagen kit, then digest PAL and COMT with Xba1 and Pst1 at 37 degrees for 2 ours in accordance with our digestion/ligation protocol.
Results: Digestion was assumed to be successful, DNA eluted in TE buffer from the Qiagen kit.
Ligate PAL and COMT digested products with Arabinose inducible vectors
Experimenter: Fisal
Aim: Ligate the PAL and COMT products to the arab inducible PSB1C3 vector with T4 ligase at a 6:1 insert to vector ratio.
Results: The ligation reaction was run overnight.
July 30th
Transformation of PAL and COMT ligations
Experimenter: Fisal
Aim: Transform PAL+ arab inducible vector and COMT+arab inducible vector into Chemically competent E.coli as per our transformation protocol.
Results: Cell were transformed and plated on Chor LB plates and grown overnight at 37 degrees C.
Colony PCR of TAL ligation to PSB1C3
Experimenter: Fisal
Aim: determine if the TAL ligation worked by means of cPCR
Results: There were no colonies on the plate, a re-attempt of the TAL→ PSB1C3 ligation is planned for the 31st.
July 31st
Re-attempt Ligation of TAL and PSB1C3
Experimenter: Fisal
Aim: Repeat the experiment from July 29th in a 20uL volume using 16uL of insert and 1 uL of vector.
Results: Ligation was run over 4 hours at room temperature. Then cells were transformed as they were on July 29th.
Test cDNA from Douglas lab to determine quality
Experimenter: Fisal
Aim: Run 5 uL of ATCCR1 containing cDNA extracted from Arabidopsis thaliana by the Douglas lab on a 1% agarose gel to determine if the sample is degraded.
Results: Success, the DNA has not been significantly degraded and should be good to use.
July
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).
Experimenter:Anna Müller
Aim:PCR minipreped DNA to amplify the DNA for digestion.
Results:PCR product confirmed on a gel.
Experimenter:Anna Müller
Aim:
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
Experimenter:Anna Müller
Aim:Additional cPCR of plates with PAL+ const.+term and COMT+const.+term.
Results:When run on a gel no bands were seen at the right bp lenght.
Experimenter:Anna Müller
Aim: Inoculate the colonies tat showed positive results for cPCR.
Results:Inoculated succesful.
September 3rd
Experimenter:Anna Müller
Aim:Miniprep the culture inoculated on September 2nd
Results:Miniprep succesful sufficent DNA
Experienter:Anna Müller
Aim:Digested with EcoRI and SpeI PAl w/arab, 4-CMH w/arab and 4-CMH w/const
Results:Digest didn't work properly, possibility of star activity.
Experimenter:Anna Müller
Aim:Colonies COMT w/arab, AttCRI w/arab were inoculated in LB containing cloramphenicol.
Results:Cultures grew up over night at 37°C.
September 4th
Experimenter:Anna Müller
Aim:PCR miniprep cleaned up
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