Molekulare biologie 2

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 ******** ************************************************