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What is the overall goal of Labs 9-12?
- 9: RE digest (HindIII and BamHI) of pAMP and pKAN plasmids, then ligation
- 10: Transformation of E. coli with recombinant DNA using classical method (CaCl2 + heat schock)
- 11: Replica plating to ID dual-resistance colonies
- 12: Purification of dual-resistance colonies and determination of which fragments are responsible
How many E. coli cells can be generated from a single cell overnight?
Why is E. coli commonly used in labs?
- Easy to work with
- Typically nonpathogenic
- Easily transformed
What is a competent cell?
- Bacteria that has been treated (made competent) to take up DNA
- *NOTE - DNA is very hydrophilic and won't normally pass through a cell membrane
- AKA induced transformation
Describe the classical method of induced transformation
- CaCL2 + heat shock
- Rapidly dividing cells have pores in their membrane
- DNA is suspended in solution of COLD Calcium ion
- DNA forced into cells by incubating cells and DNA together on ice (causing DNA to bind to surface), briefly heat shocking them (42C) (causing a few of the bound DNA molecules to be taken in by the cell), then putting them back on ice
What are the four phases of bacterial growth w/ description, and how does this apply to competency?
- Lag phase: little growth, activation of genes
- Log phase: rapid growth
- Stationary phase:
- Death phase:
- *NOTE- competence occurs late in the log phase of browth
During experiment 10 how did we verify that the E. coli was in log phase?
- Check optimal density (OD) of LB + E. coli at 550nm
- Should be between 0.3 and 0.4
Which forms of DNA are most readily transformed?
- Supercoiled DNA has TE of ~10^6
- Relaxed circular (nicked/covalently closed) has TE of ~10^4
- Linear DNA has TE of ~10^2
How is TE measured? Describe the calculation
- CFU/ug of DNA
Why did we allow a 1 hour recovery period after transformation in experiment 10?
- Kanomycin inhibits protein synthesis
- The 1 hour recovery period allow expression of Kan resistance gene product before exposure to the plate
Lab 10 - What are satellite colonies? More on AMP or KAN? why?
- Satellite colonies are not resistant, but reside in a "zone of resistance" formed by a resistant colony
- This is seen more frequently on LB-AMP, where cells live until wall replication occurs
- on LB-KAN unresistant cells will have already died
What is the ENCODE project? What results were found?
- Encyclopedia of DNA Elements
- Follow up to HGP, an effort to catalog all functional elements and regulatory elements in the human genome
- Results: 1.2% of genome codes for exons of protein-coding genes
- 80% is functional (junk DNA not a thing)
- 76% is transcribed to RNA
- 5% is conserved across all mammals (4x higher than 1.2% protein coding region)
Describe the number of genes / what they code for as determined by ENCODE
- 20687 protein coding genes
- 18400 RNA genes (no protein)
- (8800 small RNAs, 9600 large RNAs (>200nt))
- 11224 pseudogenes
What is alternative splicing?
There are ~2.53 protein coding variants per locus (range between 1 and 10)
What important RNA breakthrough was discovered with C. elegans
- sense RNA and antisense RNA worked similarly to suppress genes (no dramatic difference)
- dsRNA was 10x more potent as silencing trigger (RNA interference)
- dsRNA can be "fed" to C. elegans by causing expression in E. coli that they eat
What is miRNA? How does it work?
- microRNA: genomically encoded small non-coding RNA that help regulate gene expression
- imp. to early development
- inhibit translation of mRNA bearing complementary target sequence
- miRNA folds in on itself to form dsRNA
- dicer (RNAse) trims and cuts this dsRNA
- each strand incorporated into RISC
- RISC finds the complementary mRNA and cleaves dsRNA to ssRNA, inhibiting translation
Describe the Sanger Sequencing method (early 1980s)
- REQ - recomb plasmid, DNA pol, 1+ primer, all 4 DNA building blocks, 1 tagged ddNTP
- 4 reactions, each contains all 4 dNTPs and 1 ddNTP which is tagged with a radioactive tracer
- *NOTE- dd = dideoxy
- ddNTPs prevent further replication (chain terminator)
- Fragment to be sequenced must be cloned into a plasmid
- Billions of molecules will result in termination at various points in the sequence (A rxn 21, 24, 26; T rxn 23, 29, 33, etc)
- **NOTE - the A reaction is showing that the termination point is a T (the A was added)
- To determination the points of termination, each tube is placed on a HIGH RESOLUTION GEL (this as paper, very long)
- *NOTE - this will allow separation by DNA fragments to a single bp
- Place X-Ray film over gel overnight, silver is displaced by radioactive trackers
- Visualize film on lightbox and read upward for best results
What was the major advancement in the Sanger sequencing method in 1988 w/ name? How is data presented?
- Automated DNA sequencing4 fluorescent labels (RYBG) rather than a single radioactive tag
- Bases read by a laser/detector rather than by humans
- Only a single lane is required, and as the fragment runs off the end of the gel the laser captures the information!!!
- Data presented in an electropherogram
Describe capillary gel electrophoresis. Advantages?
- Tubes are filled w/ matrix (like a column)
- Mostly completed by robots
- Up to 384 tubes in CE by 2003
- Advantages: no gel pouring, increased # of runs/day, autoloading, robot completion
- *NOTE - no longer used, due to newer tech
What is the biggest advantage for next generation sequencing?
- No bacterial clones
- No vectors, no ligase, no transformation
- *NOTE - isolate genomic DNA, fragment the DNA, add dsDNA tag to each end
Describe emulsion PCR + pyrosequencing
- fragmented DNA has dsDNA tag added to each end
- suspended beads (single bead in water droplet) have attached primers that are complementary to the DNA tags
- PCR occurs within each droplet (only 15% successful, but others do not interfere)
- Denature DNA -> anneal to bead -> extension of DNA fragment -> denature 1 strand from beat -> repeat)
- break emulsions and deposit beads onto picotiter plate (3.6m wells, only ~1m have bead w/ single frag)
- **NOTE - each company reads the results differently
- company 1 (454 sequencing)
- mix DNA, DNA pol, ATP sulfurylase (enz), APS (subs), luciferase (enz), luciferin (subs), and apyrase (degrades nucleotides)
- 1 rxn creates ATP, other rxn creates light
- integration of nucleotide releases PPi
- PPi + APS -> ATP, ATP + luciferase -> light
- nucelotides added one at a time every 8 minutes until light is seen, then repeated
- *NOTE - apyrase is what degrades the free nucleotides in between each treatment
- Light captured by camera, height = related to # of nucleotide added
- *NOTE - only 400bp per rxn, but 1 million+ rxns
Describe bridge amplification
- similar to pyrosequencing
- amplification on "flow cell" (flat) not on a bead
- nucleotides have 4 fluorescent tags
- Flow cell imaged after each addition of 4 nucleotides
- Molecules form "bridges" as they are amplified
- software keeps track of each signal
- *NOTE - 75bp per rxn, 300 million rxns
Describe the ABI SOLiD system
- very different from other 2 next gen sequencing techniques
- 50bp per rxn, >1 billion simultaneous rxn
- high accuracy (99.94)
Describe how next generation sequencing techniques are creating breakthroughs in medicine (specific article)
- Researchers closely examined the genome of 158 people with brain malformations
- Using sensitive sequencing techniques they covered a depth (how many times each sequence is read) of 300x (more than 5x the normal depth)
- This allowed them to ID mutations that would have otherwise been dismissed as read errors!!!
- Only deep sequencing allowed them to find these mutations! (Sanger would've missed them, and it is still the "golden standard")
What is the CRISPR-Cas9 system? (general)
- CRISPR - clustered regualrly interspaced short palindromic repeats
- Cas - CRISPR associated proteins
- Cas9 is a nuclease
- First discovered in bacteria, it functions as a defense against foreign DNA (we use Type II)
- It is now a unique tool that allows insertion of DNA askjdnknfa2f
Describe the spacer-repeat construct RE: CRISPR
During invasion (viral or plasmid) a portion of the previous invader's DNA is inserted as spacers in the repeat-spacer-repeat-spacer construct
What is the function of CRISPR/Cas in bacteria?
- The CRISPR/Cas system targets foreign DNA with a short complementary ssRNA
- This localizes the Cas9 nuclease and causes a dsbreak, resulting in the silencing of that DNA sequence
- *NOTE - Protospacer Adjacent Motif (PAM) must be present for the binding of the complex
What are the 2 major breakthroughs used in CRISPR/Cas?
- The genes for crRNA and tracrRNA can be JOINED creating a single GuideRNA which is functional!!!! (simply add a loop to bind them together)
- This works in eukaryotic cells - and the ds break can be repaired (or inserted :) )
- Only the target sequence need be changed to target specific genes!
How can CRISPR/Cas be used with a library of cells?
Genes can be "knocked out" to determine function
What are the requirements for a functional plasmid (re: experiment 12)
- Have AmpR and KanR
- Have origin of replication (2 is ok)
- Hind to Hind, Bam to Bam
- Must be circular
- Limit 4 peices (large size prevents entry to E. coli)
- Same pieces cannot be "next door neighbors"
What are the two names for the "special plasmids" from experiment 12?
- Super plasmid (all 4)
- simple recombinant (only 2 required)
Why do multiple lanes appear when viewing the lanes for experiment 12? What is the important one?
- Supercoiled (will travel furthest, only one to compare)
What can't 2 fragments be "next door"?
- Complementary strands will occur both next to and across from a given strand
- this causes a loop to form, and replication is unlikely
Why do we record 3 values from the nanodrop?
- Helps to verify that we have DNA, not protein
- Concentration [ng/uL]
- 260/280 ~ 1.8 for DNA
- 260/230 ~ 2-2.2 for nucleic acids
For the C. glutamicum RE digests, why were the products passed over a column between digests?
To remove remaining RE and RE buffer that would interfere with the next digest.