MOLEKULARE BIOLOGIE 2
Praktikum 320.006
13.2.-17.2.2012
Dr Christine SiliganMag Nicole Ollinger
GENERAL STUFF
LABORREGELN
LABORMANTEL
SCHLÜSSEL FÜR SPINTS
MOLEKULARE BIOLOGIE 2 PR
DNA based methods
sdm PCR / rth PCR DNA modifying enzymes (DpnI, PNK, Ligase) E. coli transformation DNA Mini-Prep Sequence analysis
LENA
MOLEKULARE BIOLOGIE 2 PR
Protein based methods
Overexpression Purification Quality control of protein output Quantification of protein output
NICKI
WOCHENPLANDNA (Lena) Protein (Nicki)
MO PCR A sdmPCR (substitution) B rthPCR (insertion)
prepare media, solutionsgrow o/n culture
DI A DpnI, TransformationB DpnI, CleanUp, PNK, Ligation, Transformation
induction of expressionharvest
MI grow colonies lysis of E coliisolation and purification of SecA
DO DNA MiniPrepsend for sequencing
Eluate SecArun SDS-PAGEBradford assay
sequence analysis
Modifying DNA
DNA vector Features Lac Operon SecA (gene of interest)
Site directed mutagenesis aa substitution T624C LBT insertion G151LBT
Modifying DNA
DNA vector Features Lac Operon SecA (gene of interest)
Site directed mutagenesis aa substitution T624C LBT insertion G151LBT
Expression vectorpET30b SecA-cys
FEATURES
•Replication:ColE origin of replication
•Selection:KanR kanamycin resistence
•Cloning site:mcs multiple cloning site
•Expression:T7 prom T7 promotorT7term T7 terminator
•inducible expression:LacI Lac repressorLacO Lac operator
•Protein tags:His Histidine tag (6/10x)
pET30b SecA-Cys
T7term
T7prom LacO
Modifying DNA
DNA vector Features Lac Operon SecA (gene of interest)
Site directed mutagenesis aa substitution T624C LBT insertion G151LBT
lac OPERON
no lactose:negative regulation via lacI
>no transcription
lactose:binds to lacI
> transcription
Utilization of lac Operon in biotechnology:
Isopropyl-ß-thiogalactosid (IPTG) mimics allolactose not metabolised
gene of interest
gene of interest
Modifying DNA
DNA vector Features Lac Operon SecA (gene of interest)
Site directed mutagenesis aa substitution T624C LBT insertion G151LBT
SecA
Bacterial protein ATPase Dimeric Co-worker of SecYEG: (motor)
Transport of secretory proteins across membrane Integration of TM proteins into membrane
Bacterial pre-protein translocase subunit- SecA
Effrosyni Papanikou, Spyridoula Karamanou & Anastassios EconomouNature Reviews Microbiology 5, 839-851 (November 2007)
Modifying DNA
DNA vector Features Lac Operon SecA (gene of interest)
Site directed mutagenesis aa substitution T624C LBT insertion G151LBT
Site-directed Mutagenesis
Mutations in non-coding sequencesgene regulatory unitsDNA-protein interactions
Mutations in coding sequencesprotein function (enzymatic or structural)
Site-directed Mutagenesis
SecA-Cys aminoacid substitutions with cysteins
for dye-labelling
insertions of LBT motif
STUDY CONFORMATION of SecA and CONFORMATIONAL CHANGES DURING SecA ACTIVITY
Modifying DNA
DNA vector Features Lac Operon SecA (gene of interest)
Site directed mutagenesis aa substitution T624C LBT insertion G151LBT
Site-directed mutagenesisA Aminoacid substitution T624C
E A I E H P W V T K A I A N A Q5‘…gaa gcc att gaa cac ccg tgg gtg act aaa gcg att gcc aac gcc cag…3‘3‘…ctt cgg taa ctt gtg ggc acc cac tga ttt cgc taa cgg ttg cgg gtc…5‘ …622 623 624 625 626…
T624 ACTT624C TGT
>T624C S5‘P-CATTGAACACCCGTGGGTGTGTAAAGCGATTGCCAACGC-3‘OH>T624C R5‘P-GCGTTGGCAATCGCTTTACACACCCACGGGTGTTCAATG-3‘OH
x
x
>T624C R5‘P-GCGTTGGCAATCGCTTTACACACCCACGGGTGTTCAATG-3‘OH
>T624C S5‘P-CATTGAACACCCGTGGGTGTGTAAAGCGATTGCCAACGC-3‘OH
3‘
5‘
5‘
3‘
Site-directed mutagenesisA Aminoacid substitution T624C
1. sdmPCR
x
x
ELONGATION (72°C)
x
x
ANNEALING (60-68°C)
MELTING (95-98°C)
x
x
xx
xx
xx
PCRPolymerase
chain Reaction
in vitro amplification of DNA
Site-directed mutagenesisA Aminoacid substitution T624C
x
x
x
x
x
parental vectorannealing of mutant primers
elongation by PCR
1. sdmPCR
xx
x
xx
x
Site-directed mutagenesisA Aminoacid substitution T624C
x
xx
x
xx
x
2. DpnI digest !!! only dam methylated DNA is cut !!!
x
xx
x
xx
x
check for PCR product on agarose gel
Agarose gel Electrophoresis
DNA fragments separated by size
Site-directed mutagenesisA Aminoacid substitution T624C
3. Transformation of E.colix
4. Plasmid isolation5. Sequencing
xx
in vivo
amplification
of DNAPlate on LB-Kana plates
xxx
Plasmid repair
Modifying DNA
DNA vector Features Lac Operon SecA (gene of interest)
Site directed mutagenesis aa substitution T624C LBT insertion G151LBT
Site-directed mutagenesisB LBT insertion G151LBT
5‘LBT 3‘LBT
N R P L F E F L G L T V G I N L 5‘…aac cgt ccg ctg ttt gaa ttc ctt ggc ctg act gtc ggt atc aac ctg…3’3‘…ttg gca ggc gac aaa ctt aag gaa ccg gac tga cag cca tag ttg gac…5‘ …149 150 151 152 153…
Y I D T N N D G W Y E G D E L L A5‘-TAT ATT GAT ACC AAC AAC GAT GGC TGG TAT GAA GGC GAT GAA CTG CTG GCG-3‘3‘-ATA TAA CTA TGG TTG TTG CTA CCG ACC ATA CTT CCG CTA CTT GAC GAC CGC-5‘
3‘LBT
>G151LBT SGTATGAAGGCGATGAACTGCTGGCGCTGACTGTCGGTATCAACCTG
>G151LBT RCAGCCATCGTTGTTGGTATCAATATAGCCAAGGAATTCAAACAGCGG
5‘LBT
Site-directed mutagenesisB LBT insertion G151LBT
3‘LBT
5‘LBT
3‘LBT
5‘LBT
3‘LBT
5‘LBT
5‘LBT3‘LBT
1. Round-the-horn PCR
Site-directed mutagenesisB LBT insertion G151LBT
parental vectorannealing of mutant primers
elongation by PCR
1. Round-the-horn PCR
5‘LBT3‘LBT
5‘LBT 3‘LBT
Site-directed mutagenesisB LBT insertion G151LBT
2. DpnI digest !!! only dam methylated DNA is cut !!!
5‘LBT3‘LBT
check for PCR product on agarose gel
3. Phosphorylation by PNK
Site-directed mutagenesisB LBT insertion G151LBT
pp
4. Ligation
Harvey Lodish, Molecular Cell Biology, 5th edition
5‘LBT3‘LBT
LBT
Site-directed mutagenesisB LBT insertion G151LBT
5. Transformation of E.coli
Plate on LB-Kana plates
6. Plasmid isolation7. Sequencing
Harvey Lodish, Molecular Cell Biology, 5th edition
Plasmid isolationDNA MiniPrep
P2
N3
1 2 3
o/n 37°C
P1
N3
Sequencingfind suitable sequencing primer
(within ~800bp of mutation/insertion)
Send…
Sequencing ClustalW2
http://www.ebi.ac.uk/Tools/msa/clustalw2/DNA sequence alignmentFASTA format
output
>originalCGGAGATGGAAAAACTCTCCGACGAAGAACTGAAAGGGAAAACCGCAGAGTTTCGTGCACGTCTGGAAAAAGGCGAAGTGCTGGAAAATCTGATCCCGGAAGCTTTCGCCGTGGTACGTG>mutant CGGAGATGGAAAAACTCTCCGACGAAGAACTGAAAGGGAAAACCGCAGAGTTTCGTGCACGTCTGGAATGTTGCGAAGTGCTGGAAAATCTGATCCCGGAAGCTTTCGCCGTGGTACGTGoriginal CGGAGATGGAAAAACTCTCCGACGAAGAACTGAAAGGGAAAACCGCAGAGTTTCGTGCAC 60
mutant CGGAGATGGAAAAACTCTCCGACGAAGAACTGAAAGGGAAAACCGCAGAGTTTCGTGCAC 60 ************************************************************ original GTCTGGAAAAAGGCGAAGTGCTGGAAAATCTGATCCCGGAAGCTTTCGCCGTGGTACGTG 120 mutant GTCTGGAATGTTGCGAAGTGCTGGAAAATCTGATCCCGGAAGCTTTCGCCGTGGTACGTG 120 ******** ************************************************