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
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
*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))
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
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.