1. MOLEKULARE
BIOLOGIE 2
Praktikum 320.006
13.2.-17.2.2012
Dr Christine Siligan
Mag Nicole Ollinger

2. GENERAL STUFF
 LABORREGELN
 LABORMANTEL
 SCHLÜSSEL FÜR SPINTS

3. 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

4. MOLEKULARE BIOLOGIE 2 PR
 Protein based methods
 Overexpression
 Purification
 Quality control of protein output
 Quantification of protein output
NICKI

5. WOCHENPLAN
DNA (Lena) Protein (Nicki)
MO PCR A sdmPCR (substitution) prepare media, solutions
B rthPCR (insertion) grow o/n culture
DI A DpnI, Transformation induction of expression
B DpnI, CleanUp, PNK, Ligation, harvest
Transformation
MI grow colonies lysis of E coli
isolation and purification of SecA
DO DNA MiniPrep Eluate SecA
send for sequencing run SDS-PAGE
Bradford assay
sequence analysis

6. Modifying DNA
 DNA vector
 Features
 Lac Operon
 SecA (gene of interest)
 Site directed mutagenesis
 aa substitution T624C
 LBT insertion G151LBT

7. Modifying DNA
 DNA vector
 Features
 Lac Operon
 SecA (gene of interest)
 Site directed mutagenesis
 aa substitution T624C
 LBT insertion G151LBT

8. Expression vector
pET30b SecA-cys
FEATURES
T7term
•Replication:
ColE origin of replication
•Selection:
KanR kanamycin resistence
•Cloning site:
pET30b
mcs multiple cloning site
SecA-Cys •Expression:
T7 prom T7 promotor
T7term T7 terminator
LacO
•inducible expression:
T7prom
LacI Lac repressor
LacO Lac operator
•Protein tags:
His Histidine tag (6/10x)

9. Modifying DNA
 DNA vector
 Features
 Lac Operon
 SecA (gene of interest)
 Site directed mutagenesis
 aa substitution T624C
 LBT insertion G151LBT

10. lac OPERON
lactose:
binds to lacI
> transcription
no lactose:
negative regulation via lacI
>no transcription
Utilization of lac Operon gene of interest
in biotechnology:
Isopropyl-ß-thiogalactosid (IPTG)
mimics allolactose
gene of interest
not metabolised

11. Modifying DNA
 DNA vector
 Features
 Lac Operon
 SecA (gene of interest)
 Site directed mutagenesis
 aa substitution T624C
 LBT insertion G151LBT

12. SecA
 Bacterial protein
 ATPase
 Dimeric
 Co-worker of SecYEG: (motor)
 Transport of secretory proteins across membrane
 Integration of TM proteins into membrane

13. Bacterial pre-protein
translocase subunit- SecA
Effrosyni Papanikou, Spyridoula Karamanou & Anastassios Economou
Nature Reviews Microbiology 5, 839-851 (November 2007)

14. Modifying DNA
 DNA vector
 Features
 Lac Operon
 SecA (gene of interest)
 Site directed mutagenesis
 aa substitution T624C
 LBT insertion G151LBT

15. Site-directed Mutagenesis
 Mutationsin non-coding sequences
gene regulatory units
DNA-protein interactions
 Mutations in coding sequences
protein function (enzymatic or structural)

16. 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

17. Modifying DNA
 DNA vector
 Features
 Lac Operon
 SecA (gene of interest)
 Site directed mutagenesis
 aa substitution T624C
 LBT insertion G151LBT

18. Site-directed mutagenesis
A Aminoacid substitution T624C
E A I E H P W V T K A I A N A Q
5‘…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 ACT
T624C TGT
>T624C S
5‘P-CATTGAACACCCGTGGGTGTGTAAAGCGATTGCCAACGC-3‘OH
>T624C R
5‘P-GCGTTGGCAATCGCTTTACACACCCACGGGTGTTCAATG-3‘OH
>T624C S
5‘P-CATTGAACACCCGTGGGTGTGTAAAGCGATTGCCAACGC-3‘OH
x
3‘ 5‘
5‘ 3‘
x
5‘P-GCGTTGGCAATCGCTTTACACACCCACGGGTGTTCAATG-3‘OH
>T624C R

19. Site-directed mutagenesis
A Aminoacid substitution T624C
1. sdmPCR
MELTING (95-98°C) PCR
Polymerase
chain
ANNEALING (60-68°C) Reaction
x
in vitro amplification of DNA
x
ELONGATION (72°C)
x x
x
x x
x
x
x x
x

20. Site-directed mutagenesis
A Aminoacid substitution T624C
1. sdmPCR
annealing of elongation
parental vector mutant primers by PCR
x
x x
x x
x x
x x
x x

21. Site-directed mutagenesis
A Aminoacid substitution T624C
2. DpnI digest !!! only dam methylated DNA is cut !!!
x
x
x x
x x x
x
x x x
x
x x
check for PCR product on agarose gel

22. Agarose gel Electrophoresis
DNA fragments separated by size

23. Site-directed mutagenesis
A Aminoacid substitution T624C
3. Transformation of E.coli
x in vivo
x amplification
x
Plate on
of DNA
LB-Kana plates
Plasmid repair
x
x
x
4. Plasmid isolation
5. Sequencing

24. Modifying DNA
 DNA vector
 Features
 Lac Operon
 SecA (gene of interest)
 Site directed mutagenesis
 aa substitution T624C
 LBT insertion G151LBT

25. Site-directed mutagenesis
B LBT insertion G151LBT
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 A
5‘-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‘
5‘LBT 3‘LBT
>G151LBT S
GTATGAAGGCGATGAACTGCTGGCGCTGACTGTCGGTATCAACCTG
>G151LBT R
CAGCCATCGTTGTTGGTATCAATATAGCCAAGGAATTCAAACAGCGG 3‘LB
T
5‘LB
T

26. Site-directed mutagenesis
B LBT insertion G151LBT
1. Round-the-horn PCR
3‘LB
T
5‘LB
T
3‘LB
T
5‘LB
T
3‘LB
T 3‘LBT 5‘LBT
5‘LB
T

27. Site-directed mutagenesis
B LBT insertion G151LBT
1. Round-the-horn PCR
annealing of elongation
parental vector
mutant primers by PCR
5‘LBT 3‘LBT
3‘LBT 5‘LBT

28. Site-directed mutagenesis
B LBT insertion G151LBT
2. DpnI digest !!! only dam methylated DNA is cut !!!
3‘LBT 5‘LBT
check for PCR product on agarose gel

29. Site-directed mutagenesis
B LBT insertion G151LBT
3. Phosphorylation by PNK
p
p
3‘LBT 5‘LBT
LBT
4. Ligation
Harvey Lodish, Molecular Cell Biology, 5th edition

30. Site-directed mutagenesis
B LBT insertion G151LBT
5. Transformation of E.coli
Plate on
LB-Kana plates
Harvey Lodish, Molecular Cell Biology, 5th edition
6. Plasmid isolation
7. Sequencing

31. Plasmid isolation
DNA MiniPrep
P1
P2
1 2 3
N3
N3
o/n 37°C

32. Sequencing
find suitable sequencing primer
(within ~800bp of mutation/insertion)
Send…

33. Sequencing
 ClustalW2
http://www.ebi.ac.uk/Tools/msa/clustalw2/
DNA sequence alignment
FASTA format
>original
CGGAGATGGAAAAACTCTCCGACGAAGAACTGAAAGGGAAAACCGCAGAGTTT
CGTGCAC
GTCTGGAAAAAGGCGAAGTGCTGGAAAATCTGATCCCGGAAGCTTTCGCCGTG
GTACGTG
>mutant
CGGAGATGGAAAAACTCTCCGACGAAGAACTGAAAGGGAAAACCGCAGAGTTT
output
CGTGCAC
GTCTGGAATGTTGCGAAGTGCTGGAAAATCTGATCCCGGAAGCTTTCGCCGTG
original GTACGTG
CGGAGATGGAAAAACTCTCCGACGAAGAACTGAAAGGGAAAACCGCAGAGTTTCGTGCAC 60
mutant CGGAGATGGAAAAACTCTCCGACGAAGAACTGAAAGGGAAAACCGCAGAGTTTCGTGCAC 60
************************************************************
original GTCTGGAAAAAGGCGAAGTGCTGGAAAATCTGATCCCGGAAGCTTTCGCCGTGGTACGTG 120
mutant GTCTGGAATGTTGCGAAGTGCTGGAAAATCTGATCCCGGAAGCTTTCGCCGTGGTACGTG 120
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