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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 June 30th 1.3.1 Miniprep 4CL 1.4 July 1.5 July 4th 1.5.1 PCR of 4-CL 1.5.2 Retransformation COMT 1.6 July 5th 1.6.1 Digestion of 4-CL 1.6.2 Re-run PCR of 4CL 1.7 July 6th 1.7.1 Genomic DNA extraction of R.sphaeroides grown on June 28th 1.8 July 8th and 9th 1.8.1 Inoculation of COMT and miniprep 1.8.2 Received Phenylalanine ammonia lyase (PAL), 4-coumarate 3-hydroxylase (4-CMH) from iGEM HQ 1.9 July 15th 1.9.1 PCR pTet (BBa_J23118) Constitutive promoter 1.9.2 Transformation of Constitutive GFP 1.9.3 Digestion of 4-CMH and PAL from pSB1C3 1.9.4 Ligation of pTET constitutive promoter to pSB1C3 plasmid with rbs 1.10 July 16th 1.10.1 Extraction of PAL and COMT from Plasmid registry 1.10.2 Extract TAL from Rhodobacter sphaeroides Using our PCR protocol 1.11 July 17th 1.11.1 Digestion of pSB1C3 Constitutive GFP (5I) 1.11.2 Plating Streptococcus thermophilus 1.12 July 19th 1.12.1 PCR rbs(BBa_B0034; Plate 1 Well 2M) 1.12.2 PCR Streptomyces coliecolor Cinnamate: Coenzyme A Ligase (ScCCL) 1.12.3 PCR Rhodobacter sphaeroides Tyrosine Ammonia Lyase (TAL) 1.12.4 Re-attempt the TAL PCR 1.12.5 Inoculation of Streptococcus thermophilus 1.13 July 22nd 1.13.1 Purify and digest TAL PCR product 1.14 July 23rd 1.14.1 Re-Attempt PCR COMT and PAL PCR 1.14.2 Re-attempt the TAL PCR 1.14.3 Digest TAL, COMT, and PAL PCR products 1.14.4 Digestion of the pTET Constitutive promoter from pSB1C3 and rbs from PCR 1.14.5 Ligation of rbs (PCR insert) into pTET constitutive promoter (vector) & Ligation of constitutive promoter (PCR insert) into psB1C3 (vector) 1.14.6 Genomic DNA extraction of Streptococcus thermophilus 1.15 July 24th 1.15.1 Transformation of Constitutive promoter and rbs ligation & Constitutive promoter into pSB1C3 1.15.2 PCR feruoyl-CoA synthetase (Fcs) gene from Pseudomonas putida genomic DNA 1.15.3 PCR Cas9 gene from Streptococcus thermophilus genomic DNA 1.16 July 25th 1.16.1 Digestion of terminator 1.16.2 DNA extraction from Danone plain yogurt for PCR 1.17 July 26rd 1.17.1 PCR Cas9 gene from Streptococcus thermophilus genomic DNA extracted Danone yogurt 1.18 July 27th 1.18.1 Run PCR of TAL, COMT and PAL as a backup 1.18.2 Colony PCR to confirm Constitutive promoter and rbs 1.18.3 Redo PCR of fcs gene from Pseudomonas putidagenomic DNA 1.19 July 28th 1.20 July 29th 1.20.1 Run PCR of TAL, COMT and PAL as a backup 1.20.2 Ligate the Digested TAL construct to PSB1C3from July 23rd, transform ligation into E.coli 1.20.3 Digest PAL and COMT with Xba1 and Pst1 1.20.4 Ligate PAL and COMT digested products with Arabinose inducible vectors 1.21 July 30th 1.21.1 Transformation of PAL and COMT ligations 1.21.2 Colony PCR of TAL ligation to PSB1C3 1.22 July 31st 1.22.1 Re-attempt Ligation of TAL and PSB1C3 1.22.2 Test cDNA from Douglas lab to determine quality 1.23 August 2nd 1.23.1 Colony PCR of TAL+PSB1C3 plate 1.23.2 Determine if the PAL and COMT ligation to arabinose inducible promoter PSB1C3 was successful via cPCR 1.24 August 5th 1.24.1 Innoculate cPCR from August 2nd 1.24.2 Re-attempt TAL transformation 1.25 August 6th 1.25.1 Extract DNA from E.coli growing overnight from August 5th 1.26 August 7th 1.26.1 Perform a DNA extraction of COMT and PAL from cell culture 1.27 August 8th 1.27.1 Run cPCR on TAL+PSB1C3 from August 5th 1.27.2 Optimize TAL PCR 1.28 August 9th 1.28.1 Colony PCR of COMT+Terminator and 4CMH+ Terminator 1.28.2 Digest PAL/COMT + Arabinose Promoter constructs 1.28.3 Extract ATCCR1 from Arabidopsis cDNA 1.29 August 10th 1.29.1 Re-run Colony PCR of COMT+Terminator and 4CMH+ Terminator 1.29.2 Optomize ATCCR1 PCR 1.29.3 Re-digest COMT/PAL and Arabinose promoter constructs 1.29.4 Digest TAL PCR product 1.30 August 12th 1.30.1 Run ATCCR1PCR for optimization 1.30.2 Ligate PAL/COMT/ATCCR1 constructs to Terminator/PSB1C3 1.31 August 14th 1.31.1 Run Colony PCR for all ligations performed on August 12th 1.32 August 15th 1.32.1 Digest TAL PCR with Spe1 and Xba1 1.32.2 Miniprep ligation products from the 14th of August 1.33 August 19th 1.34 August 21st 1.35 August 27th 1.36 August 29th 1.37 30th August 2013 1.38 August 30th 1.39 August 31st 1.40 September 1st 1.41 September 2nd 1.42 September 3rd 1.43 September 4th 1.44 September 5th 1.45 September 6th 1.46 September 8th 1.47 September 9th 1.48 September 17th 1.49 September 18th 1.50 September 19th 1.51 September 23rd 1.52 September 24th

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 5 mL 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

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 5 mL of the LB on their respectively antibiotic. Miniprepped the 5 mL inoculation of COMT with Qiagen Miniprep Kit and eluted with 30 uL 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 5 mL 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

Plating Streptococcus thermophilus

Experimenter: Anna

Aim: Dr. Smit's lab has a strain of Streptococcus thermophilus from his lab collectioin. The strain (strain type unknown) was plated onto YPG agar plate for colony selection. Colonies were grown at 30C for 2 days.

Result: Two morphological sets of colonies were observed on the plate. Under the microscope, one set of colonies were cocci-like and the other set of colonies were rod-shaped-like. This shows that the strain was contaminated. Only the cocci types were selected.

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 60 degrees, 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 0.75 uL MgCl2 in a 50 uL reaction had the best results.

Inoculation of Streptococcus thermophilus

Experimenter: Anna

Aim: Inoculation of a colony of Streptococcus thermophilus in 5 mL YPG media and grow at 30C overnight

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 (PCR insert) into pTET constitutive promoter (vector) & Ligation of constitutive promoter (PCR insert) into psB1C3 (vector)

Experimenter: Joe

Aim: Ligate pTET constitutive promoter and rbs. Ligate constitutive promoter into pSB1C3 to clone out of pSB1A3. Incubate at room temperature overnight.

Genomic DNA extraction of Streptococcus thermophilus

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 24th

Transformation of Constitutive promoter and rbs ligation & Constitutive promoter into pSB1C3

Experimenter: Joe

Aim: Transform Constitutive promoter and rbs & Constitutive promoter into pSB1C3 ligations 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.

PCR Cas9 gene from Streptococcus thermophilus genomic DNA

Experimenter: Joe

Aim: PCR Cas9 gene from Streptococcus thermophilus genomic DNA extracted

Results: PCR failed. PCR products were ran on a 0.8% agarose gel. No bands were observed except for primer dimers.

July 25th

Digestion of terminator

Experimenter: Joe

Aim: Digest terminator with EcoRI and PstI for ligation into pSB1C3 plasmid to clone out of the pSB1A3 plasmid.

DNA extraction from Danone plain yogurt for PCR

Experimenter: Joe

Aim: DNA extraction was performed using the QuickExtract Bacterial DNA Extraction kit straight from yogurt sampels which was pelleted by centrifuge following 18% sodium citrate and 1M NaOH.

Results: Extracted DNA was diluted in TE buffer.

July 26rd

PCR Cas9 gene from Streptococcus thermophilus genomic DNA extracted Danone yogurt

Experimenter: Joe

Aim: PCR Cas9 gene from Streptococcus thermophilus genomic DNA extracted from Danone yogurt

Results: PCR failed. PCR products were ran on a 0.8% agarose gel. No bands were observed except for primer dimers.

July 27th

Run PCR of TAL, COMT and PAL as a backup

Experimenter: Joe

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.

Colony PCR to confirm Constitutive promoter and rbs

Experimenter: Joe

Aim: Colony PCR to confirm Constitutive promoter and rbs construct in transformed cells

Results: PCR successful. PCR products were ran on 0.8% agarose gel. The correct size band was observed.

Redo PCR of fcs gene from Pseudomonas putidagenomic DNA

Experimenter: Joe

Aim: PCR fcs gene from Pseudomonas putidagenomic DNA using fcs forward and reverse primers

Results: PCR products were ran on 0.8% agarose gel. PCR failed and no products were observed on the gel.

July 28th

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.

August 2nd

Colony PCR of TAL+PSB1C3 plate

Experimenter: Fisal

Aim: Determine if the ligation of TAL to PSB1C3 was successful via Colony PCR.

Results: Since the empty PSB1C3 vector reports white when a gene has been inserted using one of the Ecor1/Xba1 cut sites and one of the Spe1 and Pst1 cutsites, a quick GFP screen demonstrated which colonies have the construct we are looking for. Five white, non-green, colonies were picked for a 50uL cPCR reaction. None of the colonies were white, thus the ligation failed.

Determine if the PAL and COMT ligation to arabinose inducible promoter PSB1C3 was successful via cPCR

Experimenter: Fisal

Aim: determine if the ligation of PAL/ COMT to Arabinose inducible PSB1C3 was successful via Colony PCR.

Results: A 50uL reaction was used and a small sample was taken from each colony per tube as per our cPCR protocol. One colony from each ligation yielded the expected bands.

August 5th

Innoculate cPCR from August 2nd

Experimenter: Fisal

Aim: Re-pick all the successful colonies from cPCR ran on August 2nd and grow cells in 5 ml of LB media containing Chlor overnight.

Results: All the culture tubes had an increased optical density the next day and the negative control showed no growth.

Re-attempt TAL transformation

Experimenter: Fisal

Aim: Re-attempt the Tal transformation from July 31st but plate a 250uL of cells rather than 150uL as there were few total colonies from the July 31st plate.

Results: Transformation carried out successfully and plate was grown overnight at 37 degrees.

August 6th

Extract DNA from E.coli growing overnight from August 5th

Experimenter: Fisal

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 7th

Perform a DNA extraction of COMT and PAL from cell culture

Experimenter: Fisal

Aim: Using the Qiagen miniprep kit extract the COMT/PAL ligations to arabinose promoters.

Results: DNA extraction was successful and DNA was eluted in EB buffer.

August 8th

Run cPCR on TAL+PSB1C3 from August 5th

Experimenter: Fisal

Aim: Confirm Ligation of TAL to PSB1C3 via GFP screen then via cPCR as described before.

Results: None of the white colonies yielded the expected bands from cPCR.

Optimize TAL PCR

Experimenter: Fisal

Aim: Run several TAL PCR reactions to optimize yield, several different annealing temperatures were attempted to find the one that yields the fewest non-specfic bands the most desired product.

Results: Gel electrophoresis the best annealing temperature was 58 degrees using the same master mix as outlined above.

August 9th

Colony PCR of COMT+Terminator and 4CMH+ Terminator

Experimenter: Fisal

Aim: Run a cPCR for two of Joe’s constructs: COMT+Terminator and 4CMH+terminator. Conditions for the cPCR reaction were obtained from Joe.

Results: Colony PCR failed due to lack of template.

Digest PAL/COMT + Arabinose Promoter constructs

Experimenter: Fisal

Aim: Digest PAL/COMT + Arabinose Promoter constructs with Xba1 and Pst1 for 2 hours at 37 degrees. Cut the Vector containing the Terminator with Spe1 and Pst1 in parallel in the same conditions. Purify the cut constructs and ligate them together using T4.

Results: After Ligation was set up it was noticed that the wrong cut sites were used, the ligation would place the terminator before the promoter and gene of interest.

Extract ATCCR1 from Arabidopsis cDNA

Experimenter: Fisal

Aim: Extract ATCCR1 gene from Arabidopsis cDNA using custom primers, an annealing temperature of 60 degrees was chosen based on the thermodynamics of the custom primers. Three parallel PCR reactions were run at 60 degrees but with varying concentrations of DMSO and MgCl2 to optimize quality of bands as secondary structure was suspected to be a problem

Results: No bands were seen for two of the PCR reactions, a faint band was seen in the reaction containing 6% DMSO and 0.75 uL MgCl2 in a 50uL tube, the band was 1.2kbp as expected.

August 10th

Re-run Colony PCR of COMT+Terminator and 4CMH+ Terminator

Experimenter: Fisal

Aim: Run a cPCR for two of Joe’s constructs: COMT+Terminator and 4CMH+terminator. Conditions for the cPCR reaction were obtained from Joe.

Results: Colony PCR failed, questions were raised about the integrity of the ligation, which will be re-attempted at a later date

Optomize ATCCR1 PCR

Experimenter: Fisal

Aim: Using the previously successful mastermix from August 9th PCR of ATCCR1 was reattempted with a higher annealing temperature (63degrees) to determine if more product could be made.

Results: PCR failed, temperature was probably too high. The next ATCCR1 PCR run will be done at a lower temperature.

Re-digest COMT/PAL and Arabinose promoter constructs

Experimenter: Fisal

Aim: Ligation of the COMT/PAL +Arab oromoter constructs to terminators failed because of incorrect placement of the COMT/PAL in the terminator plasmid; the genes were ligated in front of the terminator. The terminator will be cut wit EcoR1 and Xba1, and the PAL/COMT constructs will be cut with EcoR1 and Spe1.

Results: Digests ran for 2 hours at 37 degrees and were purified using a Qiagen PCR clean up kit, DNA was eluted in ddH20.

Digest TAL PCR product

Experimenter: Fisal

Aim: Digest TAL PCR product with Xba1 and Pst1 at 37 degrees for 2 hours in preparation for ligation into PSB1C3 plasmid.

Results: Digest was completed and cleaned according to protocol. Ligation will be done on the 12th of August.

August 12th

Run ATCCR1PCR for optimization

Experimenter: Fisal

Aim: Optimize PCR for ATTCR1 using an annealing temperature of 61 degrees and 6% DMSO as described earlier.

Results: The PCR was successful with one sting band at 1.2kbp as expected.

Ligate PAL/COMT/ATCCR1 constructs to Terminator/PSB1C3

Experimenter: Fisal

Aim: Ligate the ATCCR1 construct to PSB1C3 vector; ligation should disrupt the GFP protein yielding white colonies. Digested and purified PSB1C3 was acquired from Joe. Both PAL and COMT had been demonstrated through Colony PCR to have been successfully ligated to arabinose Inducible promoters, thus ligation of these constructs to the terminator mentioned above should yield a construct that we can test expression with. A Final ligation of TAL into PSB1C3 would also be attempted.

Results: The Ligations were run at room temperature for 4 hours and were then transformed into Chemically competent cells in accordance with our transformation protocol. In addition to the ligations mentioned above ATCCR1 was also ligated into a PSB1C3 +arabinose promoter vector. As it turns out TAL has two illegal cutsites, pst1 and EcoR1, that was overlooked, some of the digests performed used EcoR1 and Pst1 thus explaining some of the difficulties encountered.

August 14th

Run Colony PCR for all ligations performed on August 12th

Experimenter: Fisal

Aim: Run colony PCR, averaging 5 -7 colonies per plate, for the following ligations: PAL-ARAB +Terminator in PSB1c3, COMT-ARAB+Terminator in PSB1C3, ATCCR1+PSB1C3, and ATCCR1+ARAB inPSB1C3.

Results: Positive results from all ligation products were observed, colonies were re picked and inoculated in 5 mL LB culture.

August 15th

Digest TAL PCR with Spe1 and Xba1

Experimenter: Fisal

Aim: Since Pst1 and EcoR1 are illegal cutsites within TAL we will use Spe1 and Xba1 for the cloning into the PSB1C3 vector before we remove the cut sited with SDM.

Results: Digest was successful and cleaned using the Qiagen PCR clean up kit.

Miniprep ligation products from the 14th of August

Experimenter: Fisal

Aim: Miniprep samples and prepare them for sequencing

Results: Samples were prepared and sent for sequencing.

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.

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.

August 30th

Experimenter: Fisal Elstone

Aim: Colony PCR TAL. PCR, digest (S/X), and Ligate AtccR1 and 4CL

to pSB1C3-1_22A-rbs. PCR 4CL.

Results: PCR/digest/ligation for AtccR1 and 4CL successful, TAL

unsuccessful.

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.

Experimenter: Fisal Elstone

Aim: PCR TAL and Gel purify, then digest (X/S), Ligate TAL pSB1C3,

digest AtccR1(X/P) and gel purify.

Results: PCR/digest of TAL successful, ligation unsuccessful. AtccR1

digest successful.

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.

Experimenter: Fisal Elstone

Aim: Colony PCR 4CL and AtccR1. Ligate/transform TAL and AtccR1 to

pSB1C3.

Results: AtccR1 and 4CL PCR success. AtccR1 transformation

successful, TAL unsuccessful.

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.

Experimenter: Fisal Elstone

Aim: Colony PCR TAL and const-AtccR1. Isolate Arab-AtccR1 and

Arab-4CL plasmids, then digest (E/S) and ligate into terminator-pSB1C3.

Results: Colony PCR of TAL successful, digest/ligation of AtccR1 and

4CL unsuccessful.

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.

Experimenter: Fisal Elstone

Aim: Miniprep Arab/const-4CL and Arab/const-AtccR1 plasmids, then

digest (S/P) and ligate into terminator-pSB1C3.

Results: Ligation was unsuccessful, but the digest was a success.

September 4th

Experimenter:Anna Müller

Aim:Miniprep culture COMT w/arab, AttCRI w/arab.

Results:DNA present.

Experimenter:Anna Müller

Aim:PCR miniprep cleaned up COMT w/arab, AttCRI w/arab.

Results:PCR succesful.

Experimenter:Anna Müller

Aim:Ligation of COMT w/const, PAL w/arab and 4-CMH w/const.

Results:see September 5th.

Experimenter:Anna Müller

Aim:Transform ligation product COMT w/const, PAL w/arab and 4-CMH w/const and plate over night on chlor.

Results:Colonies grew over night.

September 5th

Aim:cPCR of colonies transformed on September 4th.

Results:When run on a gel no bands were seen at the right bp lenght-> go backt to ligation.

Experimenter:Anna Müller

Aim:Digest of PAL w/arab, COMT w/arab and AtCCRI w/arab with EcorI and SpeI in order to ligate to terminator.

Results:Digest was successful.

Experimenter:Anna Müller

Aim:Ligation of PAL w/arab, COMT w/arab and AtCCRI w/arab to terminator.

Results:Ligation was unsuccesful see September 6th.

September 6th

Experimenter:Anna Müller

Aim:Digest of terminator with E&X

Results: Digest failed, ligation to inserts didnt work. Gel extract in the future.

September 8th

Experimenter: Fisal Elstone

Aim: Digest(E/S) previous 4CL and AtccR1, then ligate into terminatorpSB1C3.

Results: Colony PCR successful. AtccR1 ligation unsuccessful, 4CL ligation successful.

Experimenter:Anna Müller

Aim:Gel extract PAL /arab, PAL w/const, COMT w/arab, COMT w/const, 4-CMH w/arab and 4-CMH w/const.

Results:Gel extraction successful.

September 9th

Experimenter:Anna Müller

Aim:Start again from plates with promoters, reinoculate.

Results:Inoculation succesful.

September 17th

Experimenter: Cam Strachan

Aim: Set up GCMS experiments (see protocol). Optimise with EncP with converts tyrosine to cinnamic acid. Supplemt phenylalanine.

Results: High background in LB

Incubation of clones in LB creates a tone of background. Going to try different extraction methods and minimal media.

September 18th

Experimenter: Cam Strachan

Aim: Optimize GCMS experiments (see protocol).

Results: Only small amounts of the monoaromatic compounds are getting out of the cell.

Read a paper that shorter incubations actually increases the yield for this class of compounds. Also, going to try some partial lysis as the background doesn’t seem to be much of a problem now.

September 19th

Experimenter: Cam Strachan

Aim: Optimize GCMS experiments (see protocol).

Results: Seeing a nice signal for cinnamic acid now. Modified protocol and started culture for all our available parts that result in benzoic acid derivative type compounds. I don’t think the CoA intermediates are flying on the GCMS. Also, going to set up internal controls for vanillin, cinnamic acid, caffeic acid, ferulic acid and p-coumaric acid.

September 23rd

Experimenter: Cam Strachan

Aim: Run GCMS experiments (see protocol).

Results: Ran about half or our contructs. Can see many of the products, going to set up the rest of our contructs in the same way and start pulling mass spectrums off the machine to match to libraries and confirm the products. Also re-running different controls such as terminator containing plasmids ect.

September 24th

Experimenter: Cam Strachan

Aim: Run GCMS experiments (see protocol).

Results: Ran the rest of the contructs. Looks like we have examples of biosynthesis for most of the intermediates in cinnamaldehyde and vanillin synthesis. Need to get my hands on an internal control for the caffeine pathway, although some of the compound calls looks promising. Going to analyze all this data and get back to the CRISPR experiments.