4. Material and Methods
4.3 Plasmids and oligonucleotides
Table 4.4: Plasmids used in this work.
Vector Resistance Application Source
pUC19 AmpR crRNA production NEB
pRSFDuet Ka nR Protei n production Nova gen
pACYDuet Ca mR Protei n production Nova gen
pET21d Ka nR Protei n production Nova gen
petM-43 Ka nR Protei n production (Dwa rakanath, 2015)
pEC-Hi s-A Ka nR Protei n production (Dwa rakanath, 2015)
pBAD AmpR Ta rget RNA production and protein production Invi trogen
Table 4.5: Recombinant plasmids for protein production
Plasmid + Insert Description
pRSFDuet + cas7fv + cas5fv-Hi s (in MCS1) + cas6f (i n MCS2)
C-termi nal His-tagged cas5fv a nd cas7fv i n MCS1 cas6f i n MCS2
pRSFDuet + ΔAH-cas5fv, cas7fv, cas6f del etion of aa K121-Y259 of Ca s 5fv a nd insertion of 6x Gl ycine/Serine l i nker
pRSFDuet + ΔCa s7fv-loop del etion of aa 27-77 of Ca s7fv a nd insertion of 6x Gl yci ne/Serine l inker pEC-Hi s-A + SUMO-His-cas7fv Ca s 7fv wi th a 6x Hi s-SUMO-tag (N-terminal) (Dwarakanath, 2015) prs fDuet + cas7fv+cas5fv-His + cas6f-Strep C-termi nal His-tagged Ca s5fv, Ca s7fv a nd C-terminal Strep-tagged
Ca s 6f
petM-43 + cas2/3fv Ca s 2/3fv wi th N-terminal His-MBP-tag (Dwarakanath, 2015) with fra me s hift corrected
pet24d + cas3fv cas3fv wi th cas2 deleted (by Dr. Pa trick Pausch) prs fDuet + Strep-cas1 + cas2/3fv N-termi nal Strep-tagged Cas1 a nd Ca s2/3fv
pACYCDuet + Hi s-cas5fv + cas6f C-termi nal His-tagged Ca s5fv a nd Cas6f from low copy pACYCDuet vector
pRSFDuet + cas7fv One copy of cas7fv i n MCS1 (untagged)
pRSFDuet + cas7fv + cas7fv Two copi es of cas7fv (one in each MCS)
pRSFDuet + WL-neutral-cas7fv + cas5fv-His + cas6f wri s t l oops i n Ca s7fv neutralized (aa 62-67 exchanged with alanine) pRSFDuet + cas7fv-pdt + Hi s-cas5fv-pdt + cas6f For pdt-ta gged Ca s5fv a nd Ca s7fv proteins a nd untagged Ca s6f pBAD + sfgfp-cas7fv + cas5fv + cas6f For s fGFP-Cas7fv fusion protein, Ca s5fv a nd Ca s6f
pCs y_complex Type I-F Ca scade: Cs y1, Cs y2, Cs y3 a nd Cs y4 (from addgene ID 89232)
Table 4.6: Recombinant plasmids for RNA production
Plasmid + Insert Description
puc19 + wt crRNA T7 promoter + pre -crRNA with a 32 nt s pacer4 from Shewanella putrefaciens CN-32 (Dwa rakanath, 2015)
pBAD + RBS + repeat-sfgfp Repeat-tagged sfgfp RNA i ncluding RBS pBAD + RBS + sfgfp Control construct without repeat-tag on sfgfp pBAD + RBS + repeat-lacZ-α Repeat-tagged lacZ-α RNA including RBS
pBAD + repeat + sfgp + repeat pBAD + RBS + repeat-sfgfp with a dditional repeat (partial, no 5′-ha ndle) for Ca s6f binding
pBAD + repeat-sfGFP pBAD + RBS + repeat-sfgfp with RBS removed
pBAD + RBS -+ repeat-sfgp-half pBAD + RBS + repeat-sfgfp the latter half of sfgfp deleted pBAD + repeat-non-coding 500 nt compl ete non-coding region from pRSFDuet (without ATG)
cl oned in pBAD with repeat but no RBS
pBAD + Non-coding-repeat-control pBAD + repeat-non-coding with and added RBS + 7 nt s pacer sequenz + ATG
pETDuet1 + repeat + RBS+ sfgfp Repeat-tagged sfgfp RNA i ncluding RBS
110 4.3.2 Oligonucleotides
Table 4.7: Primers used for cloning
Name Sequence 5′-3′ Description
ΔAH-Ca s 5fv-invPCR-fwd ΔAH-Ca s 5fv-invPCR-rev
GGCGGCAGCGGCGGCAGCACAACGGGACCCAAA AAA
GATCATTCCAGTAAATGCAT
Invers e PCR primer to delete alpha-helical doma in of cas5fv a nd replacement with 6xGl yci ne/Serine linker
ΔCa s 7fvl oop-invPCR-fwd ΔCa s 7fvl oop-invPCR-rev
GGCGGCAGCGGCGGCAGCCTGTATATAAGTCAA AAT
CCCATTCCAATTCACTACGC
Invers e PCR primer to delete cas7fv WL-loops regi ons and replacement with
6xGl yci ne/Serine linker s
fGFP2xRepeat-invPCR-fwd
s fGFP2xRepeat-invPCR-rev
TAAGTTCACCGCCGCACAGGCGGCTTAGAAAAGC TCGAGATCTGCAGCTG
GCTGCCTTTATACAGTTCATCCATACC
Invers e PCR primer for addition of second repeat downstream of pBAD + RBS + repeat-sfgfp
Ca s 75-His-6-Strep-i nvPCR-fwd Ca s 75-His-6-Strep-i nvPCR-rev
TGGAGCCATCCGCAGTTTGAAAAATAACTCGAGT CTGGTAAAGAAACC
AAACCAAGGTACTGTAGCGG
Invers e PCR primer for addition of Strep -tag on C-termi nus of cas6f i n pRSFDuet + cas7fv + cas5fv-Hi s (in MCS1) + cas6f (i n MCS2)
l a cZ-Repeat-fwd
La cZ-Repeat-rev
GCACAGGCGGCTTAGAAAGAAGGAGATACCATG GCATGACCATGATTACGCCAAG
GAATTCCCATATGGTACCAGCTGCAGATCTCGAG CTCTATGCGGCATCAGAGCAGA
Pri mer for cl oning of l acZ-α in pBAD-Repeat, cut wi th NcoI and XhoI
mi nusRBS-fwd mi nusRBS-rev
ATGGTTAGCAAAGGTGAA GGTTTCTAAGCCGCCTGT
Pri mer for removal of RBS from pBAD + RBS + repeat-sfgfp by i nverse PCR
gfp-half-fwd gfp-half-rev
CGGTCTGATAAAACAGAATT TTCAATGCGGTTCACCAGGG
Pri mer for removal of the latter half of sfgfp by i nvers e PCR
i PCR-Ca s1-2/3-fwd i PCR-Ca s1-2/3-rev
GCGGCCGCATAATGCTTA GATTTATTCCTCATCTTC
Pri mer for removal of Ca scade genes from pRSFDuet + cas7fv + cas5fv-Hi s (in MCS1) + cas6f (i n MCS2)
Ca s 7-BamHI-fwd Ca s 7-HindIII-rev
CCAGGATCCATGCAAAAAGTAACGG CCGCAAGCTTCTATTTTGCATAAAAATACTG
Pri mer for cl oning cas7fv i n first MCS of pRSFDuet, cut by BamHI a nd HindIII Ca s 7-NdeI-fwd
Ca s 7-XhoI-rev
TACATATGCAAAAAGTAACGGG GACTCGAGCTATTTTGCATAAAAATAC
For cl oning of cas7fv i n second MCS of pRSFDuet (NdeI/XhoI)
Ca s 5-BamHI-fwd Ca s 6-NotI-rev
CAGGATCCGATGAAAATAATCATAG ATGCGGCCGCTTAAAACCAAGGTACTGTAG
For cl oning of cas5fv a nd cas6f i n pACYCDuet res tri cted with BamHI and NotI (extra G for N-termi nal His-tag)
ra ndom-pBAD-fwd ra ndom-pBAD-rev
ACGTTCACCGCCGCACAGGCGGCTTAGAAAGCAA AAAGCAAAGCACCG
CGAATTCCCATATGGTACCAGCTGCAGATCCAAC TCTTTGAACCAAGG
For cl oning of 500 nt ra ndom sequence (wi thout RBS a nd a ny ATG) from pRSFDuet ba ckbone in pBAD
i nvPCR-5-Strep-fwd i nvPCR-5-Strep-rev
CAGTTTGAAAAAAGCCAGGATCCGATGAAA CGGATGGCTCCAGCTGCTGCCCATGGTATA
Excha nge of His-tag with Strep-tag in pACYCDuet + Hi s-cas5fv + cas6f
NC-Control -fwd NC-Control -rev
CGGGAGTATGCAAAAAGCAAAGCACCG TATCTCCTTCTTTCTAAGCCGCCTGTGC
For cl oning of RBS + 7nt s pacing distance + ATG ups tream of pBAD + repeat-non-coding
111
mi nus-His-Cas5-fwd mi nus-His-Cas5-rev
TAGTAAGCGGCCGCATAATGCT AAGCTTAATGTTTGATACATAG
For remova l of C-terminal His-tag on cas5fv
N-Hi s-Cas5-fwd N-Hi s-Cas5-rev
CATCACCATCATCACCACAAAATAATCATAGAATA TG
CATGCAACCTCCTATTTTG
For a ddition of N-terminal His-tag on cas5fv
pdt1-fwd
pdt-1-revcomp
GCGGCGAACAAAAACGAAGAAAACACCAACGAA GTGCCGACCTTTATGCTGAACGCGGGCCAGGCGA ACCGCCGCCGCGTG
CACGCGGCGGCGGTTCGCCTGGCCCGCGTTCAGC ATAAAGGTCGGCACTTCGTTGGTGTTTTCTTCGTT TTTGTTCGCCGC
Synthesis von pdt#3 from (Ca meron & Col lins, 2014) for pl acement on C-terminus of cas7fv
pdt2-fwd
pdt-2-revcomp
GCAGCCAATAAGAATGAGGAGAATACGAATGAG GTTCCTACGTTCATGCTCAATGCCGGACAAGCTA ATCGTCGACGGGTC
GACCCGTCGACGATTAGCTTGTCCGGCATTGAGC ATGAACGTAGGAACCTCATTCGTATTCTCCTCATT CTTATTGGCTGC
Synthesis of pdt#3 from (Ca meron & Col lins, 2014) for pl acement on C-terminus of cas5fv (va ri ed codon sequence)
Ca s 7-wl-neutral-fwd Ca s 7-wl-neutral-rev
GCCGCCGCCGCGGCACAAGCAACTGACATTAA GCCCGTTTCATCTTTCAC
For excha nge of aa 62-66 to a lanine to neutralize wrist loops by i nverse PCR I-F-Repeat-fwd
I-F-Repeat-rev
AGAAAGAAGGAGATACC TAGCTGCCTATACGGCA
Invers e PCR to change I -Fv Repeat sequence to I-F Repeat in pBAD + repeat-non-coding pdt1+l inker-fwd
pdt1+l inker-rev
GCGGCGAACAAAAACGAAG
CGACCCCCCCCCTTTTGCATAAAAATACTG
a ddition of GSGS linker between pdt a nd cas7fv
pdt2+l inker-fwd pdt2+l inker-rev
GCAGCCAATAAGAATGAG
CGACCCCCCCCCAAGCTTAATGTTTGATAC
a ddition of GSGS linker between pdt a nd cas5fv
Table 4.8: Primers used for in vitro assays
Name Sequence 5′-3′ Description
T7s fGFP-Repeat-fwd GAAATTAATACGACTCACTATAGGGAGAGTTC ACCGCCGCACAGGCGG
Fwd Pri mer for amplification of in vitro tra ns cription template, including a T7 promotor s equence
T7-5'ha ndle-gfp-fwd GAAATTAATACGACTCACTATAGTTAGAAAGA AGGAGATAC
Fwd Pri mer for amplification of in vitro tra ns cription template, including a T7 promotor s equence and directly s tarting with processed 5′-handle (first nt C i nstead of G) T7s fGFP-Repeat-rev TTAATGGTGATGATGATGGTG Rev pri mer for a mplification of in vitro
tra ns cription template
l a cZ-probe TTGTAAAACGACGGCCAGTGAATTCGAGCTCG
GTA
Northern Blot probe for detection of l acZ-
(i n i nitial 100 nt)
Sp4-GG-tar AAGCTTGAGGGCCCAAGCCGTTATGCTAGGGT
TATAGGTTTGCGCGTCTTGCTGGGCGATAGGA CTCCCTATAGTGAGTCGTATTAGGATCC
EMSA s ubstrate i ncluding complementary s equence to crRNA spacer a nd GG-PAM
Sp1-GG-tar AAGCTTGAGGGCCCAAGCCGTTATGCTAGCAA
TGTGGTCGCGCAATTTATGATTTGGTTGAGGA CTCCCTATAGTGAGTCGTATTAGGATCC
EMSA s ubstrate i ncluding
non-compl ementary s equence to crRNA spacer a nd GG-PAM
Sp4-GG-non-target GGATCCTAATACGACTCACTATAGGGAGTCCT ATCGCCCAGCAAGACGCGCAAACCTATAACCC TAGCATAACGGCTTGGGCCCTCAAGCTT
Compl ementary s equence to Sp4-GG-tar for ds DNA constructs
112
Sp1-GG-non-target GGATCCTAATACGACTCACTATAGGGAGTCCT CAACCAAATCATAAATTGCGCGACCACATTGCT AGCATAACGGCTTGGGCCCTCAAGCTT
Compl ementary s equence to Sp1-GG-tar for ds DNA constructs
Sp4-AA-ta r AAGCTTGAGGGCCCAAGCCGTTATGCTAGGGT
TATAGGTTTGCGCGTCTTGCTGGGCGATAAAA CTCCCTATAGTGAGTCGTATTAGGATCC
EMSA s ubstrate i ncluding complementary s equence to crRNA spacer a nd a wrong PAM s equence
Sp4-AA-ntar GGATCCTAATACGACTCACTATAGGGAGTTTTA
TCGCCCAGCAAGACGCGCAAACCTATAACCCT AGCATAACGGCTTGGGCCCTCAAGCTT
Compl ementary s equence to Sp4-AA-tar for ds DNA constructs
Rl oop-mimic-tar GGTTATAGGTTTGCGCGTCTTGCTGGGCGATA GGACTCCCTATAGTGAG
EMSA s ubstrate i ncluding complementary s equence to crRNA spacer a nd GG-PAM Rl oop-mimic-ntar CTCACTATAGGGAGTCCATTATTATTT Pa rti ally complementary s equence to
Rloop-mi Rloop-mi c-tar Rl oop-Sp4-TT-tar GGTTATAGGTTTGCGCGTCTTGCTGGGCGATA
AAACTCCCTATAGTGAG
EMSA s ubstrate i ncluding complementary s equence to crRNA spacer a nd wrong PAM Rl oop-Sp4-TT-ntar CTCACTATAGGGAGTTTATTATTATTT Pa rti ally complementary s equence to
Rloop-Sp4-TT-ta r Rl oop-bubble-ntar GGATCCTAATACGACTCACTATAGGGAGTCCA
TAGCGGGTCCAAGACGCGCAAACCTATAACCC TAGCATAACGGCTTGGGCCCTCAAGCTT
Compl ementary s equence to Sp4-GG-tar with s ma ll 10 nt opening adjacent to PAM