Team Fun Guys: Lorena Christensen, Michelle Fretheim, Gabriel Martin

Our Goal
 Insert fungus Phanerochaete chrysosporium’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 carboncarbon bonds in phenolic resins.

1 ccttcgtatg taagtcgctg cggactcatg gtgtccaagc gtaggggcta acccggtgca
61 gggtcttcca cgactccatc gctatctcgc ccaagcttca gtcgcagggc aagtttgggt
121 acgcactcca gctcatgcac ggccaggtgc actgctaact ttgagttccc tcttcacagc
181 ggcggcggcg cggacggctc gatcatcacc ttctcctcga tcgagaccac gtaccacccg
241 aacatcggcc tcgacgaggt cgtcgccatc cagaagccgt tcatcgcgaa gcacggcgtc
301 acgcccggcg acttcatcgc gttcgccggt gccgtcggcg tgagcaactg cccgggcgcg
361 ccgcagatgc agttcttcct cggccgcccc gaggcgacgc aggctgcccc cgacggtctc
421 gtgcccgagc ccttccgtga gcagcagtca gcggtagagc aaacaatcga gttgctgact
481 ctccatgacc ggcagacacc atcgatcagg ttctcgctcg catgcttgat gctggcggct
541 tcgacgagat cgagactgtc tggctgctct ctgcgtgggt ggactcctcg ctggtgtcct
601 taacgctctg accctgcttc ccaccagcca ctccatcgcg gctgcgaacg acgtcgaccc
661 gaccatctcc ggcctgccgt tcgactccac ccctggccag ttcgactccc agttcttcgt
721 cgagacgcag ctccgcggta ccgcattccc tgggtgagtg gtcccgcagc agcttctgct
781 gtttccgtca ctaaaccatc attgcagcaa gaccggcatc cagggcaccg tcatgtcccc
841 gctcaagggc gagatgcgtc tgcagacgga ccacttgttc gcgcgcgact cgcgcacggc
901 g
GREEN = Exon: build with oligos
YELLOW = Exon: extract from DNA
GRAY = Intron
Join 1..9,62..118,180..436,496..574,628..753,808..901

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.

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.

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.

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

What Actually Went Down…

 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.

What Actually Went Down…

 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

What Actually Went Down…

 Sneezy (forward + biobrick extension )
GTTTCTTC GAATTCGCGG CCGCTTCTAG CCTTCGTATG TAAGTCGCTG
 Grumpy (forward without biobrick extension)
CCTTCGTATG TAAGTCGCTG
 Cowgirl (reverse + biobrick extension )
TACTAGTAG CGGCCGCTGC AGGAAGAAAC CGCCGTGCGC
GAGTCGCGCG
 Cowboy (reverse without biobrick extension)
CGCCGTGCGC GAGTCGCGCG

What Actually Went Down…

 Obtained primers in DNA form, made a working stock
(100uM).
 Ran PCR to test primer combinations:
1) Sneezy + Cowgirl
2) Grumpy + Cowboy
3) Sneezy + Cowboy
4) Grumpy + Cowgirl
 Ran each set at three different temperatures:
1. 58.0
o C
2. 51.7
o C
3. 48.0
o C

What Actually Went Down…

 Ran gel electrophoresis to verify primers worked
Lane 1: Sneezy Cowgirl 58.0
° C
Lane 2: empty
10 µl
Lane 3: Sneezy Cowgirl 51.7
° C
Lane 4: Sneezy Cowgirl 51.7
° C
Lane 5: Sneezy Cowgril 48.0
° C
Lane 6: Sneezy Cowgirl 48.0
° C
5 µl
15 µl
5 µl
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

What Actually Went Down…

 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.

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

What Actually Went Down…

 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 56 o C to lower viscosity.
However, it had the opposite effect.
 Ran RT-PCR, gel electrophoresis, and still got same results as first attempt.

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

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

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
FastRuler Ladder Low Range
Sneezy Cowgirl
15µl
15µl
15µl

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 H
2
O + 1.0uL plasmid

What Actually Went Down…

 Followed protocol from “PGem from
Promega” with adjustments.
 Plated ligations and controls:
Control 1) H
Control 2) H
2
2
O + 40uL competent cells
O + 40uL competent cells
Control 3) SOC + 50uL Competent cells
1) 3.5 uL GC to 0.5 uL plasmid
2) 3.0 uL GC to 1.0 uL plasmid on LB on LB+Xgal+Amp on LB+Xgal+Amp on LB+Xgal+Amp on LB+Xgal+Amp
3) 3.5 uL SC to 0.5 uL plasmid
4) 3.0 uL SC to 1.0 uL plasmid on LB+Xgal+Amp on LB+Xgal+Amp
5) 3.0 uL H
2
O + 1.0 uL plasmid (ligation control) on LB+Xgal+Amp

What Actually Went Down…

 Picked four white colonies each from plates #1-4.
 Added to liquid culture for miniprep of plasmid.
 Shook for one hour at 125rpm

What Actually Went Down…

 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.

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

What Actually Went Down…



What Actually Went Down…
Grumpy Cowgirl B
8 BLAST hits 
Results for Grumpy Cowgirl B: lcl|53853 GRUMPY COWGIRL B G07_GCB_T7-1_676795.seq Sequence #0 of 3 dow nloaded on Mon Dec 6 11:35:28 CST 20...(1269bp)
Graphic Summary
Distribution of 8 Blast Hits on the Query Sequence [?]
An overview of the database sequences aligned to the query sequence is shown. The score of each alignment is indicated by one of five different colors, which divides the range of scores into five groups. Multiple alignments on the same database sequence are connected by a striped line.
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.)
Descriptions
Legend for links to other resources: UniGene GEO Gene Structure Map Viewer
PubChem BioAssay
Sequences producing significant alignments:
Accession Description
Max score
Total score
Query coverage
E value
Max ident
Links
EF644562.1
Phanerochaete chrysosporium clone pchl6 lignin peroxidase isoform D (lipD) gene, partial cds
1496 1496 70%
X15599.1
M18743.1
Phanerochaete chrysosporium
LIP2 gene for lignin peroxidase
1445 1445 70%
P.chrysosporium ligninase (CKG4)
455 1109 48% mRNA, complete cds
Phanerochaete sordida ylpB gene
AB455007.1
for lignin peroxidase precursor, complete cds
233 233 43%
0.0
0.0
3e-
124
96%
95%
100%
2e-57 75%

What Actually Went Down…
Grumpy Cowgirl C
Same results
Again, our LipD gene is the top match.
Results for Grumpy Cowgirl C: lcl|63439 GRUMPY COWGIRL C H07_GCC_T7-1_676796.seq Sequence #1 of 3 dow nloaded on Mon Dec 6 11:35:28 CST 20...(1257bp)
Graphic Summary
Descriptions
Legend for links to other resources: UniGene GEO Gene Structure Map Viewer
PubChem BioAssay
Sequences producing significant alignments:
Accession Description
Max score
Total score
Query coverage
E value
Max ident
Links
EF644562.1
Phanerochaete chrysosporium clone pchl6 lignin peroxidase isoform D (lipD) gene, partial cds
1517 1517 71% 0.0 97%
X15599.1
M18743.1
Phanerochaete chrysosporium
LIP2 gene for lignin peroxidase
1465 1465 71%
P.chrysosporium ligninase (CKG4)
466 1122 48% mRNA, complete cds
AB455007.1
Phanerochaete sordida ylpB gene for lignin peroxidase precursor, complete cds
244 244 44%
0.0
2e-
127
96%
100%

What Actually Went Down…
Sneezy Cowgirl A
115 hits – all vector related
Results for Sneezy Cowgirl A: lcl|12347 SNEEZY COWGIRL A A08_SCA_T7-1_676797.seq Sequence #2 of 3 dow nloaded on Mon Dec 6 11:35:28 CST 20...(1285bp)
Graphic Summary
Distribution of 115 Blast Hits on the Query Sequence [?]
An overview of the database sequences aligned to the query sequence is shown. The score of each alignment is indicated by one of five different colors, which divides the range of scores into five groups. Multiple alignments on the same database sequence are connected by a striped line.
Descriptions
Legend for links to other resources: UniGene GEO Gene Structure Map Viewer
PubChem BioAssay
Sequences producing significant alignments:
Accession Description
Max score
Total score
Query coverage
E value
Max ident
Links
AY508731.1
Cloning vector psiLentGene
Basic, complete sequence
GU370778.1
Cloning vector pDuExAc6, complete sequence
1596
1568
1596
1666
70%
69%
0.0
0.0
97%
100%
GQ489185.1
Cloning vector pSpark II, complete sequence
EF565883.1
EF565882.1
Plant expression vector pDuExP (pDuExAn6), complete sequence
1568
1568
1568
1666
69%
69%
Plant expression vector pDuExAn1, complete sequence
1568 1666 69%
DQ917671.1
Cloning vector pGEM-WIZ, complete sequence
1568 1568 69%
0.0
0.0
0.0
0.0
97%
100%
100%
97%
DQ917670.1
Cloning vector pGEM-S1, complete sequence
1568 1568 69%
AF338824.1
Cloning vector pVLH/int(+), complete sequence
1568 1568 69%
X65309.2
Cloning vector pGEM-5Zf(-) 1568 1568 69%
AF173954.1
Cloning vector pGEM-URA3, complete sequence
1568 1568 69%
0.0
0.0
0.0
0.0
97%
97%
97%
97%

What Actually Went Down…
Sneezy Cowgirl B
Same results as the first two samples.
LipD gene is top match.
Results for Sneezy Cowgirl B: lcl|50541 SNEEZY COWGIRL B B08_SCB_T7-1_676798.seq Sequence #3 of 3 dow nloaded on Mon Dec 6 11:35:28 CST 20...(1187bp)
Graphic Summary
Distribution of 8 Blast Hits on the Query Sequence [?]
An overview of the database sequences aligned to the query sequence is shown. The score of each alignment is indicated by one of five different colors, which divides the range of scores into five groups. Multiple alignments on the same database sequence are connected by a striped line.
Descriptions
Legend for links to other resources: UniGene GEO Gene Structure Map Viewer
PubChem BioAssay
Sequences producing significant alignments:
Accession Description
Max score
Total score
Query coverage
E value
Max ident
Links
EF644562.1
Phanerochaete chrysosporium clone pchl6 lignin peroxidase isoform D (lipD) gene, partial cds
1506 1506 75% 0.0 96%
X15599.1
M18743.1
Phanerochaete chrysosporium
LIP2 gene for lignin peroxidase
1454 1454 75%
P.chrysosporium ligninase (CKG4)
466 1122 51% mRNA, complete cds
AB455007.1
Phanerochaete sordida ylpB gene for lignin peroxidase precursor, complete cds
244 244 46%
0.0
1e-
127
95%
100%
8e-61 75%

SUCCESS and FAIIIIIIIL

 Primers worked well in multiple combinations
 Extracted DNA from P. chrysosporium
 Transformation
 Ligated correct gene

SUCCESS and FAIIIIIIIL

 RNA extractions
 Steps in various protocols
 Not limiting media, therefore suppressing LiPD gene expression!!!!

Suggestions for further experiments
 RNA extraction using nutrient deprived P.
chrysosporium sample
 Clone and assemble exon fragments, as we originally planned.