lec 16-18 biotechnology and synthetic biology Flashcards

• multiple cloning sites, T vector site style, selection marker
• capable of rapid growth in inexpensive medium
• nonpathogenic (virulence genes deleted)
• capable of incorporating DNA (able produce competent cells)
• gentically stable in culture (recA deleted, why? bc recA catalyzes homologous recombination, so you dont want that to happen since cloning DNA then put in ecoli plasmid, so nothing wild happens in ecoli)
• equipped with appropriate enzymes to allow replication of the vector

ideal hosts: escherichia coli (gram - bacteria, common cloning, k12 strain, can make electrorecian or other cells), Bacillus subtilis (gram +, easily to transform, doesnt need to make more cells), Saccharomyces cerevisiae (baker yeast)

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human promoter

so if put in ecoli -> ecoli cannot recognize human promoter, so need to put human promoter in there

ecoli cannot recognize exon or intro

need to do pcr

can recognize the introns and exons

...

using in vitro techniques to alter genetic material in the laboratory

Basic techniques include:

• Restriction enzymes
• Gel electrophoresis
• Nucleic acid hybridization
• Molecular cloning
• Cloning vectors

recognize specific DNA sequences and cut DNA

Widespread among prokaryotes. Rare in eukaryotes

• Protect prokaryotes from hostile foreign DNA (e.g., viral genomes)
• Essential for in vitro DNA manipulation

Type II cleave DNA within their recognition sequence and are most useful for specific DNA manipulation

palindromes (inverted repeat sequences)

• Typically 4–8 base pairs long; EcoRI recognizes a 6-base-pair sequence

Sticky ends (allows mixing and matching) or blunt ends

invasion by foreign DNA

• Destroy foreign DNA
• Must protect their own DNA from inadvertent destruction

protect cell's DNA for restriction enzymes. Each restriction enzyme has a partner modification enzyme

• Chemically modify nucleotides in restriction recognition sequence
• Modification generally consists of methylation of DNA

separates DNA molecules based on size

• Electrophoresis uses an electrical field to separate charged molecules
• Gels are usually made of agarose, a polysaccharide
• Nucleic acids migrate through gel toward the positive electrode because of their negatively charged phosphate groups

ethidium bromide (an intercalating agent) [slips int othe DNA bases], and DNA can be visualized under UV light

different banding patterns on an agarose gel

DNA ladder

base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix

• Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a nucleic acid probe

DNA as detecting targets

RNA as detecting targets

Fluorescent In Situ Hybridization (Figure 12.5)

• Uses fluorescent probe attached to oligonucleotide

you can only amplify one strand

process:

1. heat the DNA strand to melt it, separating strands, then cool so short DNA primers can bind to the target sequence (short DNA primers bind (or "anneal") to a single-stranded DNA template at a specific temperature= anneal primers)

2. then taq polymerase extend those primers, copying DNA

3. repeating cycle, anneal primers extend 20-30 times = exponential amplification

Primers are short DNA sequences that bind to specific regions on the template DNA to initiate replication—the forward primer binds to the bottom strand and runs 5'→3', while the reverse primer binds to the top strand in reverse complement, also running 5'→3'. This image shows how primers flank the target region, with the forward primer initiating synthesis to the right and the reverse primer to the left.

start codon: ATG

stop codon: TAA

forward primer: first 18-20 bases of the coding strand

forward primer: ATGGACCAGTTCGATCAGA

reverse primer: TGCGGCGGAACTGCCG

• Phylogenetic studies
• Surveying different groups of environmental organisms • Amplifying small amounts of DNA
• Identifying a specific bacteria
• Looking for a specific gene

• Can make (c)DNA from an RNA template
• Uses the enzyme reverse transcriptase

often uses primer called oligo dt, binding in the poly-A tail found in most eukaryotic mRNA, then run PCR

• Uses fluorescent probe to monitor the amplification process

how much of the target sequence was in the starting sample

isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated

1. Isolation and fragmentation of source DNA
2. Insertion of DNA fragment into cloning vector (w sticky ends + DNA ligase)
3. Introduction of (recombinant DNA) cloned DNA into host organism - usually in ecoli (transformation)

Isolation and fragmentation of source DNA

• Source DNA can be genomic DNA, RNA, or PCR-amplified fragments
• Genomic DNA must first be restriction digested

Insertion of DNA fragment into cloning vector

• Most vectors are derived from plasmids or viruses
• DNA is generally inserted in vitro
• DNA ligase: enzyme that joins two DNA molecules

- Works with sticky or blunt ends

Introduction of cloned DNA into host organism

• Transformation is often used to get recombinant DNA into host
• Some cells will contain desired cloned gene, while other cells will have other cloned genes

• Gene library: mixture of cells containing a variety of genes

• Shotgun cloning: gene libraries made by cloning random genome fragments

Detect the correct clones

Encode proteins that are easy to detect and assay
• Examples: lacZ, luciferase, GFP genes

Promoters or coding sequences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions

cloning vectors

• Small size; easy to isolate DNA
• Independent origin of replication
• Multiple copy number; get multiple copies of cloned gene per cell
• Presence of selectable markers (usually antibiotic resistance)

chemical transformation or electroporation

• Contains ampicillin-resistance and lacZ genes
• Contains multiple cloning site (MCS) within lacZ gene

need IPTG (inducer) to make sure lacz gene is turned on in the first place

vector with foreign DNA inserted

foreign DNA inserted (cloned gene containing)

lacZ gene is inactivated by insertion of foreign DNA

• Inactivated lacZ cannot process Xgal; blue color does not develop (IPTG needed to relieve the inhibition on the lacZ promoter)

• Developed for cloning DNA products made by PCR (Figure 12.9)
• Taq adds an extra A at the 3’ ends of PCR products

• Capable of rapid growth in inexpensive medium
• Nonpathogenic (virulence genes deleted)
• Capable of incorporating DNA (able produce competent cells)
• Genetically stable in culture (recA deleted, why? bc involved in recombination, creates deletions or insertions)
• Equipped with appropriate enzymes to allow replication of the vector

ex. Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae

F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1

• F-: incapable of conjugation as donor
• lacZ: allows blue/white selection and β-galactosidase assay
• argF: blocks the production of arginine
• recA: lacks the recombinase
• endA: eliminates non-specific endonuclease activity, resulting in improved plasmid stability
• hsdR17: allows efficient transformation of DNA generated from PCR reactions *hsdR–eliminates restriction of unmethylated EcoKI sites.
• phoA: allows alkaline phosphotase assay
• supE: tRNA glutamine suppressor of amber (UAG)
• thi: requires thiamine for growth on minimal media
• gyrA: DNA gyrase mutant produces resistance to nalidixic acid
• relA: RNA is synthesized in absence of protein synthesis (relaxed phenotype) relA locus regulates the coupling between transcription and translation

vectors that are stably maintained in two or more unrelated host organisms (e.g., E. coli and B. subtilis or E. coli and yeast)

• Bacterial plasmid engineered to function in eukaryotes

• Add a eukaryotic origin of replication
• Add a centromere recognition sequence(CEN)
• Add a eukaryotic selection marker (ESM)
• Add a eukaryotic RBS
• Optimize codon usage
• Eliminate introns using RT-PCR

allow experimenter to control the expression of cloned genes (maximizing cloning production from gene)

• Based on transcriptional control
• Allow for high levels of protein expression
• Strong promoters
• Efficient operators (tight regulation)
• Effective transcription terminators are used to prevent expression of other genes on the plasmid

• In T7 expression vectors, cloned genes are placed under control of the T7 promoter
• Gene for T7 RNA polymerase (recognized by) present and under control of easily regulated system (e.g., lac)

• T7 RNA polymerase recognizes only T7 promoters
  • Transcribes only cloned genes
  • Shuts down host transcription

Use of living organisms for industrial or commercial applications

Organism whose genome has been altered

eukaryotic genes in prokaryotes (e.g., insulin)

• This is achieved by cloning the gene via mRNA

other problems

• Degradation by intracellular proteases

• Toxicity to prokaryotic host
• Formation of inclusion bodies (folds)

protein purification

genetic engineering

a growth hormone, is another widely produced hormone

• Cloned as cDNA from the mRNA
• Recombinant bovine somatotropin (rBST) is commonly used in the dairy industry; stimulates milk production in cows
  

rBST is a growth hormone for milk

Organism that contains a gene from another organism (can modify animal or plant, micro injection)

• Electroporation
• Particle gun methods (gene gun)
• Use of plasmids from bacterium Agrobacterium tumefaciens

Many successes in plant genetic engineering; several transgenic plants are in agricultural production

introduce DNA into plants

Ti plasmid, which is responsible for virulence

mobilize DNA for transfer to the plant

T-DNA

resistance to herbicides, insects, and microbial disease, as well as improved product quality

herbicide resistance

insect resistance

livestock and other animals for human consumption

Vector vaccine and Subunit vaccine

vaccine made by inserting genes from a pathogenic virus into a relatively harmless carrier virus (e.g., vaccinia virus)

contain only a specific protein or proteins from a pathogenic organism

A single vaccine that immunizes against two different diseases

• The process of isolating potentially useful novel genes from the environment without culturing the organism
• To do so, DNA (or RNA) is directly isolated from the environment and cloned into appropriate expression vectors BACs (bacterial artificial chromosomes), and the library is screened for activities of interest
• pathway engineering

The production of small metabolites by genetic engineering typically involves multiple genes that must be expressed in a coordinated manner

The process of assembling a new or improved biochemical pathway using genes from one or more organisms (e.g., indigo)

using genetic engineering to create novel
biological systems out of available parts (biobricks)

Examples

• Self-replicating synthetic bacterium
• Genetically modified E. coli that produces photographs

• recode so bacterium can only grow if supplied with a synthetic amino acid
• replaced all TAG stop codons with TAA
• express genes for an aminoacyl-tRNA synthetase that recognizes synthetic amino acid (sAA) and a tRNA with an AUC anticodon
• essential genes were modified to contain TAG codon but not affect activity