crispr system caroline vrana davidson college synthetic biology summer 2012
TRANSCRIPT
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CRISPR System
Caroline VranaDavidson College Synthetic Biology
Summer 2012
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Big Picture
• Non-promoter gene regulation
• Modular Selection Mechanism
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Full version CRISPR sequence
Yellow= BioBrick prefix and suffixBlue= leader sequence Pink= CRISPR repeatGreens= GFP target spacerReds= AmpR target spacer
GAATTCGCGGCCGCTTCTAGAGAAACAAAGAATTAGCTGATCTTTAATAATAAGGA
AATGTTACATTAAGGTTGGTGGGTTGTTTTTATGGGAAAAAATGCTTTAAGAACAAA
TGTATACTTTTAGACGGTTTATCCCCGCTGGCGCGGGGAACTCAATACTCCAATTGG
CGATGGCCCTGCCTTCGGTTTATCCCCGCTGGCGCGGGGAACTCTAAAAGTGCTCAT
CATTGGAAAACGTTCTTCGGTTTATCCCCGCTGGCGCGGGGAACTCGGTGAAGGTGA
TGCAACATACGGAAAACTTCGGTTTATCCCCGCTGGCGCGGGGAACTCCGTGTAGAT
AACTACGATACGGGAGGGCTTCGGTTTATCCCCGCTGGCGCGGGGAACTCTACTAGT
AGCGGCCGCTGCAG
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Simplified synthetic CRISPR sequence
BioBrick endsLeader SequenceCRISPR repeatGFP target spacerBamHI recognition site
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Ligation combinations
Reporter Genes• GFP
– pSB1A8– pSB4A8– pSB1C8– pSB4C8
• RFP– pSB1A8– pSB4A8– pSB1C8– pSB4C8
• CRISPR– In pSB1K8
• All ligations were successful and all in the GCAT-alog
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Oligo Assembled CRISPR Experiment Results
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Ratio of GFP fluorescence
pSB1K8 and GFP (tube 1)
pSB1K8 and GFP (tube 2)
CRISPR and GFP (tube 1)
CRISPR and GFP (tube 2)
pBad (- control) J10054 (+ control)0
0.5
1
1.5
2
2.5
Expected no green in CRISPR coloniesResults real green fluorescence
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Company Synthesized CRISPR experiments
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CRISPR in pSB1K8GFP and RFP in pSB4A8
pBad (-con-trol)
K091131 (+ green)
J04450 (+red)
C1 C2 C3 C4
-0.15
0.05
0.25
0.45
0.65
0.85
1.05
1.25
K and A plates
Expected no growthResults no growth
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CRISPR in pSB1K8 GFP and RFP in pSB4C8
pBad (-) K091131 (+) J04450 (+ red) C1 C2 C3
-0.15
0.05
0.25
0.45
0.65
0.85
1.05
K and C plates
- control
Expected no green fluorescence (only red)Results real green fluorescence
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Conclusions/Future Steps
• Company synthesized CRISPR System didn’t destroy GFP– Re-do experiment more colonies to screen
• Put into modular selection mechanism
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Background
• CRISPR– Clustered Regularly Interspaced Short Palindromic
Repeats• Functions as the prokaryotic “immune system” • Found first in E.coli in 1987• Found in 90% of archaea and 40% of bacteria
tested so far
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CRISPR process
www.wikipedia.org
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Full version CRISPR sequence
Yellow= BioBrick prefix and suffixBlue= leader sequence Pink= CRISPR repeatGreens= GFP target spacerReds= AmpR target spacer
GAATTCGCGGCCGCTTCTAGAGAAACAAAGAATTAGCTGATCTTTAATAATAAGGA
AATGTTACATTAAGGTTGGTGGGTTGTTTTTATGGGAAAAAATGCTTTAAGAACAAA
TGTATACTTTTAGACGGTTTATCCCCGCTGGCGCGGGGAACTCAATACTCCAATTGG
CGATGGCCCTGCCTTCGGTTTATCCCCGCTGGCGCGGGGAACTCTAAAAGTGCTCAT
CATTGGAAAACGTTCTTCGGTTTATCCCCGCTGGCGCGGGGAACTCGGTGAAGGTGA
TGCAACATACGGAAAACTTCGGTTTATCCCCGCTGGCGCGGGGAACTCCGTGTAGAT
AACTACGATACGGGAGGGCTTCGGTTTATCCCCGCTGGCGCGGGGAACTCTACTAGT
AGCGGCCGCTGCAG
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Problems
• Long turnaround time for synthetic CRISPR sequence
• Sent off sequence to be synthesized• In the meantime…– Simplified the sequence to only 1 target spacer and
2 CRISPR repeats– Assembling sequence on my own from
overlapping oligos
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Simplified synthetic CRISPR sequence
BioBrick endsLeader SequenceCRISPR repeatGFP target spacerBamHI recognition site
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Simplified Sequence• Includes:– BioBrick prefix and suffix– Leader sequence (in lieu of promoter)– CRISPR repeats– GFP target spacer– BamHI recognition site for expanding the
sequence in the future
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End goals
• Co-transform E.coli cells with 2 plasmids– 1. Synthetic CRISPR sequence in Kan plasmid– 2. A target plasmid (including target spacer of GFP
and/or AmpR)• Have the CRISPR plasmid destroy the target
plasmid destroying the ampicillin resistance• Assess growth (or lack of growth)
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Non-CRISPR plasmid
• Ligating different combinations of inserts/plasmids– GFP in non-AmpR plasmid– RFP in AmpR plasmid– GFP in AmpR plasmid
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Ligations/Transformations
GFP
RFP
GOIOR
OR
CRISPR
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Ligation combinations
INSERTS
• J04450 (RFP)
• K091131 (GFP)
• CRISPR sequence
• PLASMIDS
• pSB1A8
• pSB4A8
• pSB1C8
• pSB4C8
• pSB1K8
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Parts- Inserts
• GFP– K091131– pLacIQ1 + RBS + GFP + TT– Originally in pSB1A2
• RFP– J04450– pLacI + RBS + RFP + TT– Originally in pSB1A2
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Parts- Plasmids
• pSB1A8– J119043
• pSB4A8– J119048
• pSB1C8– J119045
• pSB4C8– J119049
• pSB1K8– J119046– Cloning CRISPR sequence into here
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GFP in Amp plasmids
• GFP and pSB1A8– Some larger than negative
control– Sent off MP DNA of 2
colonies to be sequence verified
– Ligation worked
• GFP and pSB4A8– Experimental wells larger
than negative control– Sent off 2 colonies to be
sequence verified– Ligation worked
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Problems with GFP
• After sequence verification of ligations-– Found 35 bp spontaneous insertion mutation– Has been documented in the promoter before – Will still work but not as bright
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RFP in pSB4A8
• Some colonies were visibly red
• Colony PCR results– Experimental DNA larger
than negative control– Sent off DNA from 2
colonies to be sequence verified
– Ligation worked
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RFP in pSB1A8
• RFP and pSB1A8
• Some colonies glowed visibly red no need to do colony PCR and sequence verification
• Ligation worked
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RFP in pSB1C8
• Cells grown from glycerol stocks of RFP and pSB1C8
• Ligation worked
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GFP and RFP in pSB4C8
• Colony PCR • Most of the colonies are larger than negative control
• Both red and green fluorescent colonies in later experiments
• Ligation worked
Neg. control
GFP
RFP
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Successful Ligations
• 8 possible combinations successfully ligated• Glycerol stocks made and located in GCAT-alog
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Problems with Cloramphenicol plasmids
• GFP and RFP in pSB4C8 • RFP in pSB1C8
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CRISPR experiment
• Oligos arrived on 7/6/12• Assembled by boiling• Ligated CRISPR sequence into pSB1K8 plasmid• Did colony PCR on 12 colonies
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Colony PCR of CRISPR sequence
• One colony seems to be the right length
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Length verification of CRISPR
• Length verification of the one colony PCR product
• Small smear of band seems to be right length (around 240)
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CRISPR Experiment
• Cotransformations• 4 experimental conditions– Only the CRISPR sequence– Only GFP in pSB4A8 and RFP in pSB4A8– Empty pSB1K8 plasmid, GFP and RFP in pSB4A8– CRISPR sequence, GFP and RFP in pSB4A8
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Co-Transformations
GFP
RFP
CRISPR
Selective Media
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Results
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Only CRISPR sequence
• Expected no growth• Result no growth
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Only GFP and RFP in pSB4A8
• Expected no growth• Results no growth
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Empty pSB1K8, GFP in pSB4A8, RFP in pSB4A8
• Expected equal amounts of green and red colonies
• Results about equal amounts of green and red colonies
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CRISPR sequence, GFP in pSB4A8, and RFP in pSB4A8
• Expected only red colonies• Results…
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Ratio of GFP fluorescence
pSB1K8 and GFP (tube 1)
pSB1K8 and GFP (tube 2)
CRISPR and GFP (tube 1)
CRISPR and GFP (tube 2)
pBad (- control) J10054 (+ control)0
0.5
1
1.5
2
2.5
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Conclusions
• The CRISPR sequence did not destroy the plasmid containing GFP
• Reason 1 nucleotide missing in the GFP target spacer when compared to the GFP gene sequence
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2nd CRISPR Sequence
• Synthesized sequence from the company came 7/18
• New Experiment– Only GFP and RFP in pSB4A8– Empty pSB1K8 plasmid, GFP and RFP in pSB4A8– CRISPR, GFP and RFP in pSB4A8– CRISPR, GFP and RFP in pSB4C8
• The CRISPR should destroy plasmids containing GFP and Ampicillin resistance
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GFP and RFP Fluorescence
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GFP and RFP in pSB4A8
pBad K091131 (+) J04450 (+ red)
NR-1 NR-2 NR-3 NR-4 R-1 R-2 R-3 R-40
1
2
3
4
5
6
7
A plates only
- control
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Empty pSB1K8 GFP and RFP in pSB4A8
pBad K091131 (+) J04450 (+ red)
NR-1 NR-2 NR-3 NR-4 R-1 R-2 R-3 R-40
0.5
1
1.5
2
2.5
3
3.5
4
4.5
- control
K and A plates
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Empty pSB1K8 GFP and RFP in pSB4C8
pBad (-) K091131 (+ green)
J04450 (+ red)
C1 C2 C3 C4 C5 C6 C7
-0.15
0.05
0.25
0.45
0.65
0.85
1.05
- control
K and C plates
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CRISPR in pSB1K8GFP and RFP in pSB4A8
pBad (-control) K091131 (+ green) J04450 (+red) C1 C2 C3 C4
-0.15
0.05
0.25
0.45
0.65
0.85
1.05
1.25
K and A plates
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CRISPR in pSB1K8 GFP and RFP in pSB4C8
pBad (-) K091131 (+) J04450 (+ red) C1 C2 C3
-0.15
0.05
0.25
0.45
0.65
0.85
1.05
K and C plates
- control
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Conclusions
• CRISPR system didn’t work– Minimal GFP fluorescence and no RFP
fluorescence
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Future Steps
• Continue working on synthetic CRISPR system• If/When the sequence works, find applications• Put CRISPR plasmid into cells destroy
something bad-ish only if the cell is making a product we want it to
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Product
stress
Modular SelectionBeneficial
E. coli
E. coli