7. Experimental part
7.2. Molecular-biological methods
7.2.1. Oligonucleotides
Oligonucleotides used in this work were purchased at Sigma Aldrich. Molecular cloning procedures are described in the manuscripts and in more detail in the following chapter.
Name Sequence 5’- 3’
RFE1 Cap2 587 1jhf for GATCCGTATC TACCAACCTC CAGGCTGGCA ACTGGGCCAA GAGCCAGGGC AACAAGAGCG AGTACCAGGCC CAAGCAGCTA CAG
RFE2 Cap2 587 1jhf rev CTGTAGCTGC TTGGGCCTGG TACTCGCTCT TGTTGCCCTG GCTCTTGGCC CAGTT-GCCAG CCTGGAGGTT GGTAGATACG
RFE3 Cap2 587 1osy for GATCCGTATC TACCAACCTC CAGGCTGGCA ACGTGGTGTT CGAGGTGAAC GGCAGAGACC TGGGCTGGGC CCAAGCAGCT ACAG
RFE4 Cap2 587 1osy rev CTGTAGCTGC TTGGGCCCAG CCCAGGTCTC TGCCGTTCAC CTCGAACACC ACGTT-GCCAG CCTGGAGGTT GGTAGATACG
RFE5 Cap2 587 EDA for GATCCGTATC TACCAACCTC CAGGCTCTGT ACAACCCCAC CACCTACCAG ATGGACGCCC AAGCAGCTAC AG
RFE6 Cap2 587 EDA rev CTGTAGCTGC TTGGGCGTCC ATCTGGTAGG TGGTGGGGT TGTACAGAGC CTG-GAGGTTG GTAGATACG
RFE51 NheI-Start-EGF-for AAAAAGCTAG CATGAACAGC GACAGCGAGT GCCC
RFE52 GGSG_EGF_rev AGATCCTCCA CCAGATCCAC CACCCCTCAG CTCCCACCAC TTCAGG
64
RFE53 Linker-mCherry-for GGTGGTGGAT CTGGTGGAGG ATCTATGGTG TCCAAGGGCG AAGAGG
RFE54 mCherryXhoI-Rev AAAACTCGAG TCAGTGGTGG TGGTGGTGGT GCTTGTACAG CTCATCCATG CCGC RFE56 Cap6/9 SwaI for ATAATAATTT AAATCAGGTA TGGCTGCCG
RFE57 RFC10 rev ATAATACTGC AGCGGCCGCT ACTA RFE58 EcoRV MscIdel for GCGAGCTCGA TATCAAATTA CGCC RFE59 CAT MscIdel rev CGTGGCTAAT ATGGACAACT TCTTCG RFE60 CAT MscIdel for CGAAGAAGTT GTCCATATTA GCCACG RFE61 EcoRI MscIdel rev GCCGCGAATT CCAGAAATCA
RFE62 Cap9 453 His for CCGGATTAGG CCATCATCAT CATCATCATA GCCAGAATCA ACAAACGCTA AAATTCAGTG TGGCCGGACC CAGCAACATG GCT
RFE63 Cap9 453 His rev GTACAGCCAT GTTGCTGGGT CCGGCCACAC TGAATTTTAG CGTTTGTTGA TTCTGGCTAT GATGATGATG ATGATGGCCT AAT
RFE64 Cap9 587 His for CCACAAACCA CCAGAGTGCC CAATTAGGCC ATCATCATCA TCATCATAGC GCACAGGCGC AGACCGGCTG GGTTCAAAACC AAGG
RFE65 Cap9 587 His rev GATCCCTTGG TTTTGAACCC AGCCGGTCTG CGCCTGTGCG CTATGATGAT GATGATGATG GCCTAATTGG GCACTCTGGT GGTTTGTGG
RFE66 Cap9 587 EDA for CCACAAACCA CCAGAGTGCC CAACTGTACA ACCCCACCAC CTACCAGATGG AC-GCACAGGC GCAGACCGGC TGGGTTCAAA ACCAAGG
RFE67 Cap9 587 EDA rev GATCCCTTGG TTTTGAACCC AGCCGGTCTG CGCCTGTGCG TCCATCTGGT AG-GTGGTGGG GTTGTACAGT TGGGCACTCT GGTGGTTTGT GG
RFE68 Cap9 587 1jhf for CCACAAACCA CCAGAGTGCC CAATGGGCCA AGAGCCAGGG CAACAAGAGC GAG-TACCAGG CACAGGCGCA GACCGGCTGG GTTCAAAACCA AGG
RFE69 Cap9 587 1jhf rev GATCCCTTGG TTTTGAACCC AGCCGGTCTG CGCCTGTGCCT GGTACTCGCTC TTGTTGCCCT GGCTCTTGGC CCATTGGGCA CTCTGGTGGT TTGTGG
RFE70 Cap9 587 1osy for CCACAAACCA CCAGAGTGCC CAAGTGGTGT TCGAGGTGAA CGGCAGAGAC CTGGGCTGGG CACAGGCGCA GACCGGCTGG GTTCAAAACC AAGG
RFE71 Cap9 587 1osy rev GATCCCTTGG TTTTGAACCC AGCCGGTCTG CGCCTGTGCC CAGCCCAGGT CTCTGCCGTT ACCTCGAACAC CACTTGGGCA CTCTGGTGGT TTGTGG
RFE72 CAT BspEI del for CATACGAAAT TCCGGGTGAG CATTC RFE73 CAT BspEI del rev GAATGCTCAC CCGGAATTTC GTATG
RFE78 Cap6 453His for CCGGATTAGG CCATCATCAT CATCATCATA GCAGTGCCCA AAACAAGGAC TTGCTGTTTA GCCGGGGGTC TCCAGCTGGC ATGTCT
RFE79 Cap6 453His rev GTACAGACAT GCCAGCTGGA GACCCCCGGC TAAACAGCAA GTCCTTGTTT TGGG-CACTGC TATGATGATG ATGATGATGG CCTAAT
RFE80 Cap6 587His for CCACCGAAAG ATTTGGGACT GTGGCAGTCA ATCTCCAGAG CAGCAGCTTA GGCCATCATC ATCATCATCA TAGCACG
RFE81 Cap6 587His rev GATCCGTGCT ATGATGATGA TGATGATGGC CTAAGCTGCT GCTCTGGAGA TTGACTGCCA CAGTCCCAAA TCTTTCGGTG G
RFE82 Cap6 5871jhf for CCACCGAAAG ATTTGGGACT GTGGCAGTCA ATCTCCAGAG CAGCAGCTGG GCCAAGAGCC AGGGCAACAA GAGCGAGTAC CAGACG
RFE83 Cap6 5871jhf rev GATCCGTCTG GTACTCGCTC TTGTTGCCCT GGCTCTTGGC CCAGCTGCTG CTCTG-GAGAT TGACTGCCAC AGTCCCAAAT CTTTCGGTGG
RFE84 Cap6 5871osy for CCACCGAAAG ATTTGGGACT GTGGCAGTCA ATCTCCAGAG CAGCAGCGTG GTGTTCGAGG TGAACGGCAG AGACCTGGGC TGGACG
RFE85 Cap6 5871osy rev GATCCGTCCA GCCCAGGTCT CTGCCGTTCA CCTCGAACAC CACGCTGCTG CTCTG-GAGAT TGACTGCCAC AGTCCCAAAT CTTTCGGTGG
65 7.2.2. General cloning procedures
Modifications of the 453 and 587 loop were carried out using the unique restriction enzyme sites for all AAV serotypes. The Rep2Cap2 plasmid was already equipped with unique SspI and SalI (453 region) as well as PvuII and BamHI (587 region). Plasmids for serotype 6 and 9 were created starting from the Rep2Cap2 plasmid. Cap6 and Cap9 were ordered as string syntheses at GeneArt (ThermoFisher Scientific). Before introduction into the pSB1C3_001-Rep2 plasmid, restrictions sites, enabling loop modifications, had to be eliminated from the plasmid backbone. By PCR reac-tion MscI (RFE60+RFE61) and BspEI (RFE72+RFE73) restricreac-tion sites were eliminated from the CAT marker region. The oligonucleotide pair RFE56 and RFE57 was used to amplify Cap6 and Cap9 from strings before ligation with the Rep2 plasmid which was opened with SwaI and PstI.
Introduction of peptide motifs in loop regions is possible using the now unique restriction sites BspEI/BsrGI (453 region) and MscI/BamHI (587 region). Complementary oligonucleotides with overlapping ends between those recognition sites were designed, that match the overhangs gener-ated during a restriction digest. After phosphorylation (T4 PNK) and annealing of complementary oligonucleotides a subsequent ligation into the digested and dephosphorylated (AnP) vector can occur.
Table 3: Standard protocols for common cloning procedures. Incubation temperatures or detailed procedures are chosen according to the manufacturer’s instructions.
Restriction digest, preparative CutSmart buffer 5 µl
Enzyme 1 µl
Plasmid 1 µg
Water until 50 µl
Phusion PCR
GC buffer 10 µl
10 mM dNTPs 1 µl
DNA template 100 ng 10 µM Primer for/rev each 2.5 µl DMSO (final 3%) 0.5 µl Phusion HF polymerase 0.5 µl
Water to 50 µl
T4 DNA ligase
Ligase buffer 2 µl T4 DNA Ligase (5 U/µl) 1 µl
Vector DNA 100 ng
Insert DNA 1:1 to 5:1 molar ratio over vector
Water to 20 µl
pJET blunt protocol
(CloneJet PCR cloning Kit #K1231) 2× Reaction buffer 5 µl
PCR product 4 µl
pJET1.2 0.5 µl
T4 ligase 0.5µl
Polymerase chain reactions (PCRs) were carried out using Phusion polymerase (NEB) with the standard protocol described by the manufacturer. All restriction and cloning enzymes were pur-chased at NEB and used according to manufacturer’s instructions. For ligation reactions a T4 ligase from ThermoFisher Scientific was applied with the provided manual. Plasmids were transformed into chemically competent E. coli DH5α cells, plated on LB agar plates with the corresponding antibiotic and incubated at 37 °C overnight. Plasmid DNA was isolated from bacterial colonies using the NucleoSpin Plasmid Kit from Macherey-Nagel. Cloning success was verified via se-quencing at the Bielefeld University Sese-quencing Core Facility. Larger quantities of plasmid DNA for transient transfections were generated according to the instructions for the NucleoBond Xtra Midi Kit from Macherey-Nagel.
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7.2.3. Plasmids
Table 4: List of plasmids used during this work.
Number Name Source
pZMB0088 pHelper Agilent
pZMB0091 pSB1C3_001_pCMV_DARPinE01_mli_VP23 _453_587koHis iGEM pZMB0154 pSB1C3_001_Rep_VP123_453_587ko_p5tataless iGEM pZMB0155 pSB1C3_001_Rep_VP13_453_587ko_p5tataless iGEM
pZMB0165 pSB1C3_001_Rep_VP123_453_587ko1jhf_p5tataless RFE master thesis pZMB0166 pSB1C3_001_Rep_VP123_453_587ko1osy_p5tataless RFE master thesis pZMB0193 pSB1C3_001_Rep_VP123_453_587koEDA_p5tataless RFE master thesis pZMB0246 pSB1C3_001_CMV_VP1up_NLS_mVenus_VP23_453_587koHis iGEM
pZMB0307 pSB1C3_001_pCMV_Kozak_VP23_453_587wt KSC
pZMB0490 pET21a-EGF-mCherry-His6 This work
pZMB0493 pSB1C3_001_FUCA1 This work
pZMB0494 pSB1C3_001_FUCA1_T2A_EGFP This work
pZMB0495 pSB1C3_FUCA1_T2A_EGFP_hGHpA This work
pZMB0496 pSB1C3_CMV_FUCA1_T2A_EGFP_hGHpA This work
pZMB0497 pUC19bb_ITR_EXS_CMV_FUCA1_T2A_EGFP_hGHpA This work pZMB0522 pUC19bb_ITR_EXS_pCMV_mVenus_hGHpolyA PBO pZMB0550 pSB1C3_002_RepCap2_VP123_453_587wt_p5tataless This work pZMB0551 pSB1C3_002_RepCap9_VP123_453_587_p5 This work pZMB0552 pSB1C3_002_RepCap9_VP123_453His_587_p5 This work pZMB0553 pSB1C3_002_RepCap9_VP123_453_587His_p5 This work pZMB0554 pSB1C3_002_RepCap9_VP123_453_587EDA_p5 This work pZMB0555 pSB1C3_002_RepCap9_VP123_453_5871jhf_p5 This work pZMB0556 pSB1C3_002_RepCap9_VP123_453_5871osy_p5 This work pZMB0573 pSB1C3_002_RepCap6_VP123_453_587wt This work pZMB0574 pSB1C3_002_RepCap6_VP123_453_587wt_p5 This work pZMB0575 pSB1C3_002_RepCap6_VP123_453_587wt_p5tataless This work pZMB0576 pSB1C3_002_Rep(PVC)Cap9_VP123_453_587_p5 This work pZMB0592 pSB1C3_002_Rep2Cap6_453His_587_p5 This work pZMB0593 pSB1C3_002_Rep2Cap6_453_587His_p5 This work pZMB0594 pSB1C3_002_Rep2Cap6_453_5871jhf_p5 This work pZMB0595 pSB1C3_002_Rep2Cap6_453_5871osy_p5 This work