March

3/9: Began settling on main projects and planning a general overview of schedule for the coming months.

May

5/4: Narrowed down project idea.

5/11: Began researching chemicals involved in chocolate taste/smell.

5/18: Continued researching chocolate taste/smell chemicals and began researching possible pathways

5/28: Continued discussion on gene pathways. Finalized furaneol and phenylethylamine DNA sequences, looked more into concrete pathways for 2,3,5-trimethylpyrazine and came to the conclusion that 3-ethyl-2,5-dimethylpyrazine would be a more viable option.

June

6/4: Finalized genes and assembled list of gene sequences. Decided to use Lactococcus lactis over E. coli for actual expression of genes due to its relevance to the food industry.

6/8: Ordered gene constructs.

6/25: Designed and ordered primers for amplifying the pNZ8148 vector.

6/29: Ran PCR to amplify the vector in preparation for HiFi assembly for each gene insert. Gel showed 2 bands of unknown length and no visible ladder. Ladder was not vortexed before being inserted into gel, likely causing the lack of visible bands in the ladder. Annealing temperature was set to 63 C, but 3’ ends of primers have a melting temperature of around 60 C, so we planned to repeat at lower annealing temperature.

July

7/6: Re-ran PCR on all constructs at lower annealing temperature (55 C) and achieved successful vector linearization for Xmt-Mxmt-Dxmt, PtPDC, and PgPDC.

7/13: Ran HiFi assembly on all genes, including those which did not show a positive band from 7/6 gel electrophoresis as the lack of bands may have been due to a gel loading error. Found nanodrop concentrations and performed Dpn1 digestion in order to remove template DNA from PCR products.

7/14: Repeated PCR and achieved successful amplification for all constructs. The ladder showed up faint using 5 uL, so we plan to increase load to 10 uL for future gels.

7/20: Performed Dpn1 digestion and Hifi assembly on all PCR products.

7/27: Transformed Hifi assembly products into DH5a.

August

8/3: After unsuccessful transformations of Hifi assembly products, began a new round of PCR, Dpn1 digestion, and column purification on all gene constructs. Transformed pNZ8148 into DH5a E. coli to verify that the lack of growth is not because of issues with chloramphenicol plates (positive controls worked, but were grown on ampicillin plates). Also plated the rest of the transformed DH5a E. coli with higher amounts of bacteria.

8/10: Created new chloramphenicol plates at a chloramphenicol concentration of 10 ng/uL instead of the default 35 ng/uL. Transformed 7/20 Hifi assembly products into MC1061 E. coli and performed Hifi assembly on 8/3 PCR products. Began PCR reaction for EGFP construct. Gel showed a negative band.

8/16: Re-plated leftover transformed MC1061 cells using 200 uL instead of 100 uL of cell/SOC mixture. Transformation of 8/10 Hifi assembly products into MC1061 and DH5a, ensuring a heat shock time of 45 seconds for MC1061 cells instead of 30 seconds.

8/17: Growth on DH5a plates from 8/16 transformations. Colony PCR on 10 colonies from the PgPDC plate produced negative bands.

8/21: Ran colony PCR on 8/16 transformation growth and found 5/7 bands positive for Xmt-Mxmt-Dxmt and 1/7 bands positive for TDH-KBL (All others were negative). Transformed 8/10 Hifi assembly products into MC1061. Performed Dpn1 digestion and column purification on EGFP PCR products. After column purification, concentration of PCR product ended up around 8.5 ng/uL (not high enough for Hifi assembly), so sample was lyophilized for future resuspension in lower volume to ensure higher concentration. Also plated more leftover MC1061 transformed cells.

8/23: Found growth on plates from 8/21 and ran Colony PCR on colonies from original DH5a plates (for gene constructs SlPDC, PtPDC, and PgPDC, performed colony PCR on 20 colonies. For gene construct FBA-TPI, performed colony PCR on 5 colonies from DH5a and 15 colonies from MC1061 plates).

8/24: Ran colony PCR on gene constructs Xmt-Mxmt-Dxmt and TDH-LBL from 8/23 MC1061 cell growth. Made cell master plate for FBA-TPI MC1061 plate. Made liquid cultures for 4 FBA-TPI positive colonies from MC1061 plate in preparation for column purification. Performed a new round of PCR on all PDC gene constructs (as no successful amplification in E. coli had resulted for any of them). Transformed leftover Hifi assembly products of PDC genes, trying both 1 uL and 5 uL of DNA for transformations.

8/27: Performed Dpn1 treatment and column purification on all PDC PCR products. Resulting concentrations were too small, so PCR products were lyophilized for future resuspension in lower volume to ensure higher concentration for Hifi assembly. Performed plasmid prep for FBA-TPI, but resulting concentration was way too low (~5 ng/uL), likely because the E. coli was cultured in 1 mL media instead of 5 mL and we let E coli grow for 3 days until starting plasmid prep. Performed Hifi assembly on EGFP and transformed into MC1061 E. coli using 2 different DNA amounts (2 uL and 3 uL).

8/31: Plates from 8/27 transformation of assembled EGFP did not grow. Resuspended lyophilized PDC genes to ensure DNA higher concentration than previously.

September

9/1: Performed plasmid prep on Xmt-Mxmt-Dxmt, TDH-KBL, and FBA-TPI from positive MC1061 colonies and recorded Nanodrop concentrations.

9/7: Made electrocompetent L. lactis cells. Made and filter sterilized wash/electroporation solution from 0.5 M sucrose solution and 10% glycerol. Because of strange pink color of the presumed L. lactis cells, it was concluded that the cells were likely contamination. Performed Hifi assembly on EGFP construct. Transformed all genes into MC1061 E. coli. Performed colony PCR on genes to verify primer binding (since some purified plasmid samples sent for sequencing were unable to bind to primer).

9/12: Re-transformed all gene constructs after finding limited growth on plates from 9/7 round of transformations. Ran PCR on purified plasmid samples in order to re-confirm their successful assembly. Gel showed positive bands for inserts, suggesting successful assembly.

9/14: Found no growth from 9/12 transformations. Made new 100ug/ml ampicillin plates.

9/18: Recorded OD600 of L. Lactis growth in 2 different mediums: LB and M17. Made competent L. Lactis cells. Made 8 liquid cultures for Xmt-Mxmt-Dxmt, TDH-KBL, and FBA-TPI from original plates in preparation for plasmid prep.

9/21: Performed plasmid prep on FBA-TPI. Performed electroporation of Xmt-Mxmt-Dxmt, TDH-KBL, and FBA-TPI into L. lactis.

9/27: Found no growth from 9/21 electroporations. Received fixed plasmid (without RepC mutation) and performed PCR on fixed plasmid.

9/28: Performed Dpn1 digest on 9/27 PCR products.