1. Site-Directed Mutagenesis

2. 01. GFP & GFP Derivatives
02. Site-Directed Mutagenesis
03. pET Expression System

3. GFP&GFP Derivatives
Wild type GFP
Green fluorescent protein (GFP) traditionally refers to the protein first isolated from the jellyfish Aequorea victoria.
GFP is a protein which exhibits bright green fluorescence when exposed to blue light.
The protein composed of 238 amino acids (26.9kDa).
The protein sequence and its resultant three-dimensional folding structure into the 11-strand β-barrel, is most likely crucial to the formation of the chromophore and its bioluminscene.

4. GFP&GFP Derivatives
Wild type GFP
The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum.

5. Formation of the GFP Chromopore
GFP&GFP Derivatives
Formation of the GFP Chromopore
GFP has the ability to exhibit intrinsic fluorescence thanks to three amino acids that cyclise (Ser65-Tyr66-Gly67) and then undergo an oxidation step during a complex maturation process.

6. Limitations with Wild-type GFP
GFP&GFP Derivatives
Limitations with Wild-type GFP
Wild-type GFP can be excited by light at two different absorption peaks, which the optimal excitation one is by UV light. This is problematic if one wants to use GFP in live cells, since UV radiation can damage them.
Wild-type GFP folds poorly at 37°C, so it limits its usefulness for in vivo experiments in the lab.
It can take up to four hours to fold GFP correctly

7. GFP&GFP Derivatives
GFP Derivatives
A diversity of mutants have been derived from GFP to emit different wavelengths of light; these include :
cyan fluorescent protein (CFP)
yellow fluorescent protein (YFP)
blue fluorescent protein (BFP)

8. GFP&GFP Derivatives
GFP Derivatives
Changes in the amino acids in the fluorophore region, as well as in amino acids downstream from the fluorophore, result in changes in the color the fluorophore emits.

9. GFP Derivatives GFP&GFP Derivatives

10. Mutagenesis Methods of mutagenesis Site-Directed Mutagenesis Random
DNA recombination
Targeted
Random change in residues or exhaustive enumeration of amino acid substitutions within selected sites
Point mutations

11. Approaches for directed mutagenesis
Site-Directed Mutagenesis
Approaches for directed mutagenesis
-> Site-directed mutagenesis
-> Point mutations in particular known area
Result -> library of wild-type and mutated DNA (site-specific)
not really a library -> just 2 species
-> Random mutagenesis
-> Point mutations in all areas within DNA of interest
Result -> library of wild-type and mutated DNA (random)
a real library -> many variants -> screening
If methods efficient -> mostly mutated DNA

12. Point mutations (Directed mutagenesis)
Site-Directed Mutagenesis
Point mutations (Directed mutagenesis)
Point mutations (directed mutagenesis)
Substitution: change of one nucleotide (i.e. A-> C)
Insertion: gaining one additional nucleotide
Deletion: loss of one nucleotide

13. Site-Directed mutagenesis
PCR-based methods
PCR
Substitution
PCR
Deletion
Site-directed mutagenesis by traditional PCR.
Primers incorporating the desired base changes are used in PCR. As the primers are extended, the mutation is created in the resulting amplicon.
PCR
Terminal Substitution

14. Polymerase Chain Reaction (PCR)
Site-Directed Mutagenesis
Polymerase Chain Reaction (PCR)
An in vitro method for the enzymatic synthesis of specific DNA sequences
Requires
Two specific oligonucleotide primers
DNA polymerase
dNTPs
Template DNA

15. Site-Directed mutagenesis

16. Site-Directed mutagenesis
Primer design guidelines
The mutagenic oligonucleotide primers for use in this protocol must be designed individually according to the desired mutation. The following considerations should be made for designing mutagenic primers
Both of the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid.
Primers should be between 25 and 45 bases in length, with a melting temperature (Tm) of ≥78 ℃.
The desired mutation (deletion or insertion) should be in the middle of the primers with ~10-15 base of correct sequence on both sides.
The primers optically should have a minimum GC content of 40 % and should terminate in one or more C or G bases

17. Site-Directed mutagenesis
Primer design example
DNA sequence : AT->GC
5’-agaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtg-3’
3’-TCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCAC-5’
5’-agaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtg-3’
3' CAGGGTTAAGAACAACT ATCTACCACTACAATTAC 5‘ CG GC
5' GTCCCAATTCTTGTTGA TAGATGGTGATGTTAATG 3'
3’-TCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCAC-5’
GC content: 40.54%         Melting temp: 75.1 °C         Mismatched bases: 2 Length: 37 bp                 Mutation: Substitution 5' flanking region: 17 bp    3' flanking region: 18 bp    

18. Protein Expression Strategy
pET Expression system
Protein Expression Strategy

19. Genetic Elements Essential for Expression
pET Expression system
Genetic Elements Essential for Expression
Expression cassette
Promoter
Regulator binding site
Ribosome binding site (RBS)
Multiple cloning site (MCS)
Transcription terminator
Antibiotic resistance gene
Origin of replication
Other control gene

20. pET Expression system
pET system
The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli.
Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell.
T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell’s resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction.

21. pET system Promoter & Expression genes
pET Expression system
pET system
Promoter & Expression genes
lac promoter (host) – T7 RNA polymerase
T7 promoter (vector) – Target gene
Regulator binding site (lac O)
Control gene (lac I)
T7 RNA polymerase inhibitor (T7 lysozyme)

22. Working Mechanism of pET vector system
pET Expression system
Working Mechanism of pET vector system

23. Experimental Scheme EGFP → EYFP 2014.04.01 PCR 2014.04.08
Products are checked by electrophoresis
PCR
Treatment of Dpn I
Making
calcium competent cells
(E. coli DH5a, E. coli BL21 (De3))
Transformation into E. coli DH5a
Plasmid preparation
Transformation into E. coli BL21 (De3)
Expression of EYFP

24. Report Materials & Methods
EGFP와 EYFP nucleotide, protein sequence 찾아서 비교하기
203번 amino acid를 치환하기 위한 Primer design (GC content, Tm, base pair…)
PCR method 정리

25. Q&A