Molecular techniques - tools for regulation

plamids, cloning, regulation

Plamids

• extra chromosomal DNA elements used in DNA exchange

  • thorugh transformation

Plasmid maintenance

• 1. high copy number

• 2. partitioning genes

• 3. resistance markers

• 4. addiction molecules

Plasmid key features

1. origin of replication and an area to clone

2. want to have many plasmids per bacteria (40-50)

   1. allows daughter cell to have plasmids

High copy number

• Want bacterial cell to have many plasmid copies to transfer to daughter cells

  • substantially increases chances

  • forces bacteria to replicate plasmid

Partitioning genes

• Help carry individual plasmids with chromosome during division

Resistance markers

• often utilize antibiotic resitance

  • keep growing only bacteria with plasmid

  • can also use metals

Addiction modules

• having a poion encoded on plasmid; plasmid also has antidote

• 1. poison is long lasting while antidote is short lasting

• 2. each daughter cell gets equal dose of poison

• 3. cell must replicate plasmid to survive (produce antidote)

Restrictive modification

• Protective mechnism against harmful DNA integration (bacteriophage DNA)

  • use of exonucleases

Guest lecture: Yersinia Pestis

• Plasmids determine virulence

  • chromosome encoded virulence

• Lipoproteins/proteins assiciated with adhesion

• T3SS delivers YOPs to trigger apoptosis, inhibit phagocytosis, and block cytokine production

• F1 encoded by caf gene: immune evasion

exonucleases (function)

• cleave (retrict) foregin DNA at specific sequences

  • DNA considered foregin if it has not been properly modifed (methylation)

Types of restriction endonucleases

1. Class I: Multisubunit enzymes with specific requirments (ATP/Mg/S-adenosylmethionine) and cut DNA at a site distant from sequence (~100 basepairs)

2. Class II: only require Mg and typically cut within palidromic sequence Ex) EcoRI

3. Class III: Requires ATP and cleaves DNA at sites 25-27bp away

Class I restriction endonucleases (mechnism)

• Act like molecular motors that cut ~100bp away from the recognition site [ cut cannot be predicted]

  • translocates DNA at a rate of 400bp/sec (reason for ATP)

  • Methylase and nuclease are part of the same complex (reason for SAM)

Class II restriction endonucleases (mechnism)

• cuts within or near the (usually palidromic) recognition sequence (only Mg needed for cut)

  • shift cut away from site always at same distance

Class III restriction endonucleases (mechnism)

• Wors similar to type I but cuts closer to recogistion site (predictable)

  • requires ATP but not SAM

  • currently no commerically avaible type IIIs

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Dr. Olsons favorite cloning vector

pBluescript II KS (-)

• lac site, ampicillin resistance, 3.0 kb long

Cloning with restriction enzymes

1. Extract DNA

2. cut DNA with restriction enxyme

3. cut plasmid with same restriction enzyme

4. Incubate the DNA fragments with plasmid→ add enzyme DNA ligase (closes nicks in dna seq)

5. Transform the ligated products into e. coli

6. grow abcteria on plates containing antibiotic (whichever resistance your vector encodes for)

7. Screen the colonies for inserted DNA

Incubating DNA with Plasmid

• Allow the DNA to sit for hours so that ligase can connect all the DNA fragments

  • Both plasmid and DNA have similar ends (lots of plasmids/DNA go back together)

Screening colonies (why?)

Important because cell should have plasmid but unknown if insert in the plasmid correctly

Blue/White screening

• uses lacZ gene product (b-glactosidase) to determine if piece of DNA has been clonded into the multiple cloning site (MCS) of the plasmid

  • good colonies = white (lacz imparied)

  • addx-gal and if cut, blue forms

Ways to measure regulation

1. measure enzyme activity

2. activity of a reporter

3. mRNA concentration

   1. northern blot

   2. qRT-PCR

4. Measure protein level

   1. SDS page

   2. Western Blot/ ELISA

Reporter Genes

1. Beta- galactosidase

2. GFP

beta-glactosidase assay

• fuse the promoter of the gene of interest to lacZ (on plamis or in the chromosome)

• Grow cells under conditions you want to compare

• add colir reagent to cells (x-gal=blue ONPG=yellow)

• Measure color - sepctrophotometer: more color, more expression

Trp operon - regulation with lacZ

• (-) tryptophan = activation of trpEDCBA genes to make trp

• (+) tryptophan = reppresion of trpEDCBA genes

• TrpE is an anthanilate synthase

• how can we tell if trp genes are on

• measure anthanilate synthase too hard

• OR replease the trpE gene with lacZ - B-gal → easy to measure

grow cells (±) trptophan→ add ONPG or x-gal→measure amount of product with spectrophotometer

• + trp - absorbance reading - 10 units

• - trp - absorbance reading - 10,000 units

Green flurorecent protein

• GPF naturally fluorescent protein from jelly fish aequoreus victoria

• good reporter since you dont have to fix cells or add color reagnet to see expression

GFP reporter

• substrate is light so you can shine UV light on live vells and check for fluorescence

Direct mRNA measurement

• Northern blot - uses DNA:RNA hybrid to measure mRNA level

• RT PCR: two steps

  • 1st - make DNA copy of mRNA with reverse transcriptase

  • 2nd - amplify DNA copy by PCR

Northern Blot

• DNA:RNA hybrid to measure mRNA level

  • labeled DNA sequence to probe for RNA

  • only tells you presence of mRNA, not quantity

RT-PCR - steps

Process of making DNA copy from mRNA

1. use reverse transcriptase and a DNA primer to produce complementary DNA strand

2. add taq polymerase, second primers, dNTPs, and heat

3. Initiate PCR process and camera measures fluorescence

Quantification with PCR process

• reaction takes place in the presence of sybr green dye that fluoresces when bound to dsDNA

  • therocycler measures the flurorescence

  • computer graphs according to cycle #

• the more mRNA, the more DNA, higher fluorescnece

Quantification 10th round

detecttion limit; acts as threshold for camera

Cycle # vs mRNA values

The more mRNA you start with, the earlier the increase in fluroreence

in early cycles → very little DNA

Measure protein: western blot

• measures the amount of protein expressed

• Disadvantage: uses an antibody against protein

• advanatge: sometimes the amount of protein expressed is not entirely dependent on the amount of mRNA expressed

  • translational regulation

  • protein turnover rates are not always equal

SDS-PAGE

• First step of western blot: proteins are separated by size via SDS gel electrophoresis

• each protein is denatured and give negative charge by treatment with detergent SDS

• Smaller proteins travel further down the gel

Antibody detection w/ western blot

• want to find protein of interest out of all bands

• must use a antibody that only recognizes your protein

• Antibody gets detected by blotting

  • radioactive (film)

  • enzyme reaction (color)

  • Enzyme reaction (light, film)

Genome wide regulation

• gene arrays: used to measure the amount of mRNA from all the genes in an organims

• RNA seq: uses next generation DNA sequencing to determine transcriptome

• 2-D gels: used to measure the amount of all the proteins expressed

• LC-MS-MS: mass spectrophy identifies proteins

Gene arrays

• each gene of a sequences organims is ficed to a slide in a n ordered array

• The array is then proped with cDNA library of mRNA from cell (two conditions)

• upregualated (green)

• down regulated (red)

Gene array steps

• Experiment with two conditions

  • sample a and b

• Isolate all RNA from both samples

• generate cDNA from this bc - block cDNAse

• label probes - two different colors

• take probes and hybridize to an array

• mixed color - 50/50 each

not quantitative

RNA seq work flow

• better than gene array for transcriptomics

• 1. isolate total RNA

• 2. eukaryote vs prokaryote

  • easier in eukaryote

  • need to get rid of rRNA(will be overexpressed and get very little signal for mRNA)→

  • if eukaryote → polyadenylate their rRNA (3’end they add As) → put over oligoDT column (has DNA and mRNA will hybridze to TTT (poly ADT)-….→ postive selection

  • Prokaryote dont so remove rRNA with a kit →…negative selection

• removed rRNA and have mostly mRNA left

• will fragment it between 300nt and 700nt - illumina

• hexmer primers and cDNA synthesis

• Ends replacement, adapter → have primers for sequencing rxn

• sequence all mRNA

• how cDNA is islated that corrsepnd to mRNA

RNA seq protocol

sample rRNA—amplify it→cDNA—make fragments→ have reads—reads→ just count the reads

Counts to expression levels

RPKM for gene i = 10^9 x(ci/l’N)

tag count 1,000,000/ total number of tags kilobase of transcript

ci# tags (reads) length * total tags

Proteins and proteomics

• gene arrays can tell about transcriptional regulation, but there are lots of conditions by which you want to know about the levels of proteins in the cell

• They study of all proteins in a cell is called proteomics

• Advantages: Detects non-transcriptional control, can be cheaper

• Disadavnatges: cheap methods miss low abundance proteins

2D gel electrophoresis

1. separates by charges (depends on pH)

   1. all proteins hav particular pH at which they have no charge, which is called their isoelectric point

2. separates by size (SDS Page)

acid proteins on one side, basic proteins on the other. Large at top and small at bottom

1st Dimension - isoelectric focusing

• charge on a protein depends on the pH

• All proteins have a particular pH at whihc they have no charge, which is called their isoelectric point

• pH<pI→ positive charge

• pH>pI → negative charge

• pH=pI → no charge

• would have a strip with gel and have immoblized pH graident

  • proteins with positve migrate toward negative electrode until they lose enough protons to lose charge

  • Each stops at its isoelectric point → no charge

• Put this gel on top on regular SDS-page gel

  • run a current and separate by size

Tryptic digest

• trypsin cuts proteins after R and K residues [Arginine and Lysine]

• Excise the spot, treat the spot with trypsin to cut it up.

• Weigh the fragments by Mass spectroscopy

Eliminate the gel with LC-MS workflow

• proteins goes through tryptic digestion to create peptide mixture

  • LC/MS and MS/MS spectroscopy creates a gas flow which leads to an elution time

  • full MS spectrum leads to quantification

  • fragmetn selected peptide leads to spectrum levels that have peptide identifications

Peptides→ digest them→ liquid chromatography→Dual mass spec→ get protein spectra→ figure out what peptides each one of these

Guest Lecture: bordetella pertussis

• Key virulence: exotoxins

• Hfq is a post-transcriptional regularotr that b. pertussis has

  • regulates abundanec of protien

• When iron starved, must adapt

• Changes in BvgAS two component system →regulates turning off and on virulent, avirulent, and intermediate genes

• Regulation of Hfq increaed abundance of Ptx (toxin) in iorn staved conditions

• Hfg decreased abundance of two fimbrae proteins (fim2 and fimC) and one adhesion protein

• Hfq regulates abundance of FhaB in low iron conditions

• Basically hfq is involved in post-transcriptional regulation of certain B. pertussis virulence factors in iron starved conditions

• Hfq helps with tolerance to oxidative stress that was originally induced by low iron levels

Computer put the sizes together

• computer program determines the zies of all proteolytic fragments from the predicted genome sequence

• computer compares experimental mass spec to predicted mass spec from database

  • then identifies porteins