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Plasmids and oligonucleotides

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