Transcript E. coli

RT-PCR
Obtain the internal region of cDNA
1. Mix crude RNA or poly(A) RNA, cDNA primer (one of
the amplification primers in 7.)
2. Heat at 650C for 3-15 min.
3. Cool on ice
4. Add RT and RT buffer, 420C (370C to 550C, depending
on the composition of primer and RNA) for 1 hr.
5. Dilute and phenol extraction
6. Ethanol precipitation
7. Mix cDNA, amplification primers, dNTPs, 940C for 2
min.
8. Add Taq polymerase, run PCR.
9. Analyze PCR products on agarose or nondenaturing
PAGE.
To obtain full-length cDNA.
(BD Clontech) 3’-RACE, and 5’-RACE
amplification kits to obtain upstream and
downstream cDNA, and clone.
Chapter 23: Discovery and analysis of
different genes or differentially expressed
genes
With two bacteria,
(BD-clontech) PCR-select Genomic substraction kit
1. Construct gene library with genes markedly different
in the two bacteria (eg. presence or absence of
pathogenicity islands).
2. Then use genomic DNA probes (radiolabelling by
random-priming) of both bacteria to check the
colonies of the library.
To generate differentially expressed library
(BD-clontech) PCR-Select cDNA substraction kit
1. Prepare tester cDNA and driver cDNA (in excess)
2. Add adaptors
3. Two rounds of hybridization
4. PCR
To find the differentially expressed genes from the
differentially expressed library
(BD-clontech) PCR-Select Differential screening kit
Colonies hybridized with tester cDNA probe, driver
cDNA probe, substracted cDNA probe, and nonsubstracted probe.
(Colonies with +,－,+, － are the differentially expressed
genes; Colonies with,－, －, +, － are very likely the
differentially expressed genes)
(BD-clontech) Delta differential display kit.
Using sets of T primers and ten arbitrary primers that
found by computer analysis of the coding regions of
more than 200 mRNAs
1. Prepare first-strand ss cDNA of two samples
2. PCR with α32P-dATP, 3 low-stringency cycles and
22-25 high-stringency cycles.
3. 5% non-denaturing PAGE and autoradiography
4. Clone the fragments different with the two samples.
AFLP (amplification fragment length
polymorphisms) (unit 23.5)
1.
2.
3.
4.
5.
6.
Genomic DNAs or cDNAs of many samples.
Two rounds of PCR
Denaturing sequencing gel
Eluting the ssDNA fragment
Polymerization by klenow
Clone
From BRL
Chapter 19: Analysis of protein interaction
In vivo two-hybrid systems
(BD) Matchmaker yeast 2-hybrid system 3
- Use the binding and activation domains of
yeast Gal4 transcriptional factor
(B42 is an artificial Gal4 activation domain).
- NLS (nuclear localization sequence) is added to
both domains.
- Reporter gene can be on plasmid or chromosome.
- Binding sequence upstream of reporter gene: GAL4uas
Other systems using SOS box (or LexAop) as binding
sequence and LexA protein as BD, to avoid the
endogenous GAL4uas and Gal4 protein in yeast.
(BD) Matchmaker mammalian 2-hybrid kit
Use mammalian vectors
pM: contains p53 as BD
pVP16: contains vp16 as AD
pGSCAT : reporter plasmid, protein interaction
induce CAT expression in cells.
pGSSEAP : reporter plasmid, interaction induce
SEAP expression in culture medium.
Co-transfect cells with pM, pVP16, and pGSCAT
/ pGSSEAP, incubate for 48 hrs, and assay for CAT or
SEAP activity.
(Stratagene) BacterioMatch 2-hybrid system
Clone bait gene to the C-terminus of cI in
pBT, and the interacting target gene to the Nterminus of the α subunit of RNA polymerase in
pTRG .
Reporter genes in an operon for histidine
prototrophy and streptomycin resistance.
Many pre-made mammalian cDNA libraries in
pTRG are available.
(Stratagene) Cytotrap 2-hybrid system
For detection of protein-protein interactions occurring on the
cell membrane.
- pMyr vector: contains BD which will be myristilated and
binds (inserts) to cell membrane.
- pSos vector: contain human homolog of CDC25 (hSos),
encoding GTP exchanging factor (GEF), as
AD.
(GEF will bind Ras and activate Ras signal pathway to
allow yeast cells to grow.)
- cdc25H yeast: a cdc25ts yeast mutant. Cells grow
normally at 250C, but cannot grow at 370C.
Protein interaction will allow the cdc25H yeast to grow at
370C.
Affinity purification of proteins binding to GST fusion proteins
(GST pulldown assay)
1. Prepare E. coli cell extract (competitor, to remove background).
2. Mix 35S-radiolabelled test protein and E. coli cell extract.
Incubate 15 min on ice.
3. Add GST or GST-protein bound to agarose beads to cell extract
in an eppendorf tube, Mix.
4. Add 3. to 2. Incubate 1-2 hr at 40C.
5. Centrifuge to remove supernatant. Wash beads with binding
buffer.
6. Add SDS sample buffer. Boil for 5 min, SDS-PAGE and
autoradiography.
Preparation of E. coli cell extract by sonication.
Surface Plasmon Resonance (SPR) using BIAcore
(detection)
1. Assemble NTA chips in BIAcore.
2. Inject 10ul of 1 mM NaOH
3. Inject His-tag protein
4. Inject target protein
5. Measure dissociation and association rates
6. Use 200 mM immidazole to wash the His-tag protein
and the His-tag protein complex.
Can use amine-coupling kit (BIAcore) to coating target
proteins on the nude chip.
Detection of protein-protein interaction by co-precipitation.
1. Prepare whole cell extract
2. Incubate with antibody against protein 1
3. Incubate with protein-A sepharose
4. Centrifuge to collect beads
5. Wash pellet
6. Dissociate proteins from protein A-sepharose by adding
SDS sample buffer and boiling.
7. Supernatant for SDS-PAGE
8. Immunoblot with antibody against protein 2.
9. Re-probe with antibody against protein 2.
Identification of protein interactions by far western
analysis.
Protein samples immobilized on PVDF membranes,
and probed with
A) an 35S-labelled non-antibody protein, or
B) an unlabelled protein probe, and further detected
with a probe-specific antibody.