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Phenolic Resin Remediation using LiP Gene from P. chrysosporium in E. coli

Our goal was to insert the LiP gene from P. chrysosporium into E. coli to enable the bacteria to break down phenolic resin. Despite challenges with primer construction and RNA extraction, we made progress in culturing the fungus and extracting DNA.

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Phenolic Resin Remediation using LiP Gene from P. chrysosporium in E. coli

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  1. Remediation of Phenolic Resin Using E. coli Containing LiP Gene from P. chrysosporium Team Fun Guys: Lorena Christensen, Michelle Fretheim, Gabriel Martin

  2. Our Goal • Insert fungus Phanerochaetechrysosporium’s Lignin Peroxidase (LiPD) gene into E. coli to see if we could enable the bacteria to break down phenolic resin (plastic). • Enzymes coded for by LiPD are able to break carbon-carbon bonds in phenolic resins.

  3. Phanerochaetechrysosporium’s Lignin Peroxidase (LiPD) gene

  4. Original Plan • Build smaller exon segments using oligonucleotides. • Use primers to extract larger exons from DNA. • Run PCR for both to amplify gene’s exons. • Ligate exons. • Transform E. coli with LiPD gene from fungus P. chrysosporium. • Plate on phenolic resin to observe remediation if successful.

  5. Modified Plan • Due to difficulty of constructing primer sets and removing introns, we decided to attempt RNA extraction instead. • Discovered there are no existing biobricks for LipD gene. We would have to build and integrate these ourselves.

  6. What Actually Went Down…(summary… details on the following slides) • Cultured P. chrysosporium • DNA extraction from P. chrysosporium • Built and ordered primers - one set with biobrick extension, one without. • PCR with primers • Attempted RNA extraction from P. chrysosporium (FAIL) • Ligation of LiPD gene into pGEM-T Easy Vector • Transformation of E. coli with vector containing LiPD gene • Miniprep of plasmid, digestion using EcoR1 • Sequenced select samples from digestion to see if LiPD gene was present.

  7. What Actually Went Down… • Cultured P. chrysosporium • Liquid cultures of mycelium using slant culture obtained from Dr. Bumpus. • Incubated at room temperature for five days in an agar suspension.

  8. What Actually Went Down… • DNA Extraction from P. chrysosporium • Filtered mycelial mats from liquid agar, let dry. • Divided into two samples as a precautionary measure: S1 and S2. • Protocol used: “Rapid procedure for the extraction of DNA from fungal spores and mycelia,” by John J. Weiland. http://www.fgsc.net/fgn44/weiland.html • Deviation from protocol: During extraction initial mixture was too viscous, so increased volume of extraction buffer added.

  9. What Actually Went Down… • DNA Extraction from P. chrysosporium • Ran agarose gel electrophoresis to verify if DNA was present  DNA!!!! Sample #1 18 µl Sample #1 6 µl Ladder Sample #2 6 µl Sample #2 18 µl

  10. What Actually Went Down… • Built and Ordered Primers • Sneezy(forward + biobrickextension) GTTTCTTC GAATTCGCGG CCGCTTCTAG CCTTCGTATG TAAGTCGCTG • Grumpy (forward without biobrick extension) CCTTCGTATG TAAGTCGCTG • Cowgirl (reverse + biobrickextension) TACTAGTAG CGGCCGCTGC AGGAAGAAAC CGCCGTGCGC GAGTCGCGCG • Cowboy (reverse without biobrick extension) CGCCGTGCGC GAGTCGCGCG

  11. What Actually Went Down… • PCR With Primers • Obtained primers in DNA form, made a working stock (100uM). • Ran PCR to test primer combinations: • Sneezy + Cowgirl • Grumpy + Cowboy • Sneezy + Cowboy • Grumpy + Cowgirl • Ran each set at three different temperatures: • 58.0o C • 51.7o C • 48.0o C

  12. What Actually Went Down… • PCR With Primers • Ran gel electrophoresis to verify primers worked Lane 1: Sneezy Cowgirl 58.0°C 10 µl Lane 2: empty Lane 3: Sneezy Cowgirl 51.7°C 5 µl Lane 4: Sneezy Cowgirl 51.7°C 15 µl Lane 5: SneezyCowgril 48.0°C 5 µl Lane 6: Sneezy Cowgirl 48.0°C 15 µl Lane 7: Grumpy Cowboy 58.0°C 10 µl Lane 8: empty Lane 9: Grumpy Cowboy 51.7°C 5 µl Lane 10: Grumpy Cowboy 51.7°C 15 µl Lane 11: Grumpy Cowboy 48.0°C 5 µl Lane 12: Grumpy Cowboy 48.0°C 15 µl Lane 13: Ladder (FastRuler Low Range) 15 µl Lane 14: Control 20 µl Lane 15: Sneezy Cowboy 58.0°C 10 µl Lane 16: empty Lane 17: Sneezy Cowboy 51.7°C 5 µl Lane 18: Sneezy Cowboy 51.7°C 15 µl Lane 19: Sneezy Cowboy 48.0°C 5 µl Lane 20: Sneezy Cowboy 48.0°C 15 µl Lane 21: Grumpy Cowgirl 58.0°C 10 µl Lane 22: empty Lane 23: Grumpy Cowgirl 51.7°C 5 µl Lane 24: Grumpy Cowgirl 51.7°C 15 µl Lane 25: Grumpy Cowgirl 48.0°C 5 µl Lane 26: Grumpy Cowgirl 48.0°C 15 µl

  13. What Actually Went Down… • Attempted RNA Extraction from P. chrysosporium (failtastic) • Used “Rneasy Plant MiniKit” protocol. • Ground mycelium with liquid nitrogen. • Purified sample for Reverse Transcriptase – PCR. • Ran RT-PCR following protocol from “QIAGEN One Step RT PCR Kit” • Ran gel electrophoresis.

  14. What Actually Went Down… Gel results: Bands in all four lanes at 900 bp– the size of our gene INCLUDING introns. RNA extraction failed. Sneezy Cowgirl RT-PCR 15 µl Sneezy Cowgirl PCR only (control) 15 µl FastRuler Ladder Low Range 15 µl Grumpy Cowgirl RT-PCR 15 µl Grumpy Cowgirl PCR only (control) 15 µl

  15. What Actually Went Down… • RNA Extraction round two • Followed same protocol with the following adjustments: • Added DNase to bind up excess DNA • Used RNase free materials to not destroy any RNA present • Heated extraction at 56oC to lower viscosity. However, it had the opposite effect. • Ran RT-PCR, gel electrophoresis, and still got same results as first attempt.

  16. What Actually Went Down… Sneezy Cowgirl PCR (control) 15 µl Sneezy Cowgirl RT-PCR 15 µl FastRuler Ladder Low Range 15 µl Grumpy Cowgirl PCR (control) 15 µl Grumpy Cowgirl RT-PCR 15 µl

  17. What Actually Went Down… • Ligation of LiPD gene into pGEM-T Easy Vector • Following protocol from “QIAquick PCR Purification Kit 50”, we purified DNA obtained from failed RT-PCR. • Purified two samples • Grumpy+Cowgirl • Sneezy+Cowgirl • Ran gel electrophoresis

  18. What Actually Went Down… • Gel used to determine concentration of DNA in our samples (needed for ligation). • Compared to ladder containing 60ng DNA/ 15µl, samples are estimated to contain 30ng DNA/ 15µl Grumpy Cowgirl 15µl FastRuler Ladder Low Range 15µl Sneezy Cowgirl 15µl

  19. What Actually Went Down… • Ligation of LiPD gene into pGEM-T Easy Vector • Did five ligation combinations: 1) Grumpy+Cowgirl 1:1 ratio 3.5uL sample + 0.5uL plasmid 2) Grumpy+Cowgirl 3.0uL sample + 1.0uL plasmid 3) Sneezy+Cowgirl 1:1 ratio 3.5uL sample + 0.5uL plasmid 4) Sneezy+Cowgirl 3.0uL sample + 1.0uL plasmid 5) Control 3.0uL H2O + 1.0uL plasmid

  20. What Actually Went Down… • Transformation of E. coli with vector containing LiPD gene • Followed protocol from “PGem from Promega” with adjustments. • Plated ligations and controls: Control 1) H2O + 40uL competent cells on LB Control 2) H2O + 40uL competent cells on LB+Xgal+Amp Control 3) SOC + 50uL Competent cells on LB+Xgal+Amp • 3.5 uL GC to 0.5 uL plasmid on LB+Xgal+Amp • 3.0 uL GC to 1.0 uL plasmid on LB+Xgal+Amp • 3.5 uL SC to 0.5 uL plasmid on LB+Xgal+Amp • 3.0 uL SC to 1.0 uL plasmid on LB+Xgal+Amp • 3.0 uL H2O + 1.0 uL plasmid (ligation control) on LB+Xgal+Amp

  21. What Actually Went Down… • Transformation of E. coli with vector containing LiPD gene • Picked four white colonies each from plates #1-4. • Added to liquid culture for miniprep of plasmid. • Shook for one hour at 125rpm

  22. What Actually Went Down… • Miniprep of plasmid, digestion using EcoR1 • Used protocol “GeneJet Plasmid MiniPrep Kit”. • Cut plasmid with EcoR1 using protocol “Fast Digestion DNA” from class packet. • Ran a gel electrophoresis to verify sizes of fragments.

  23. What Actually Went Down… • Miniprep of plasmid, digestion using EcoR1 gel results Lane 1: 3.0 Grumpy Cowgirl 19 µl Lane 2: 3.5 Grumpy Cowgirl 19µl Lane 3: 3.5 Grumpy Cowgirl 19µl Lane 4: 3.5 Grumpy Cowgirl 19µl Lane 5: FastRuler Ladder Low Range 19µl Lane 6: 3.0 Sneezy Cowgirl 19µl Lane 7: 3.0 Sneezy Cowgirl 19µl Lane 8: 3.0 Sneezy Cowgirl 19µl Lane 9: 3.5 Sneezy Cowgirl 19µl Lane 10: 3.5 Sneezy Cowgirl 19µl Lane 11: 3.5 Sneezy Cowgirl 19µl

  24. What Actually Went Down… • Sent four samples (lanes 3,4,6,7) from digestion to be sequenced. • Ran the sequences with NCBI BLAST to see if our gene was present.

  25. What Actually Went Down… Grumpy Cowgirl B 8 BLAST hits  Red indicates an excellent alignment score for 5 of the 8 hits. Pink indicates a very good score. Descriptions of sequences with significant alignment. The top hit IS our gene of interest. (Note: E-values indicate the likelihood that this hit is unrelated to our query.)

  26. What Actually Went Down… Grumpy Cowgirl C Same results Again, our LipD gene is the top match.

  27. What Actually Went Down… Sneezy Cowgirl A 115 hits – all vector related

  28. What Actually Went Down… Sneezy Cowgirl B Same results as the first two samples. LipD gene is top match.

  29. SUCCESS and FAIIIIIIIL • Successes: • Primers worked well in multiple combinations • Extracted DNA from P. chrysosporium • Transformation • Ligated correct gene

  30. SUCCESS and FAIIIIIIIL • Failures: • RNA extractions • Steps in various protocols • Not limiting media, therefore suppressing LiPD gene expression!!!!

  31. Suggestions for further experiments • RNA extraction using nutrient deprived P. chrysosporiumsample • Clone and assemble exon fragments, as we originally planned.

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