Genetic Engineering Final

Sense Codons

Encode an amino acid

Wobble Hypothesis

Third base can change and still code for the same amino acid

Codons are written as they appear in the mRNA

5’ to 3’

mRNA and tRNA pair in an

Anticodon fashion

Quaternary Structure

Two or more polypeptide chains associate

Tertiary Structure

Secondary structure folds

The amount of codons present in the genetic code

64 codons

tRNA Charging

Amino acid binding to tRNA

The amount of different aminoacyl-tRNA synthetase in a cell

20

Shine-Dalgarno Consesus

Sequence in bacterial gene recognized by the small unit ribosome

Kozak Sequence

Facilitates the identification of the start codon in eukaryotic cells

Initiation of Translation

Only involves the small ribosomal subunit, forms fmet-tRNA

Ectopic gene expression

Tool used to analyze gene function and protein science

Gene expression constructs

cDNAs and ORFs

cDNAs and ORFs

Used to overexpress a protein for changes in phenotype, localization, or isolation and purification

cDNAs and ORFs are derived from

Native mRNA that can facilitate gene expression and over-expression assays

ORFs are created from cDNA by

Removing the untranslated regions leaving just the protein coding region for expression

Overexpression of the Tomato ERF-36 gene alters growth related traits by

Regulating stomata number and carbon assimilation

RNAi/PTGS

Double stranded RNA that regulates the expression of protein-coding genes

Natural mechanism for sequence-specific gene silencing

RNAi

Resistant to parasites and pathogens

RNAi

RNAi Gene Silencing

Result of nucleolytic degradation of the targeted mRNA by the RNase H enzyme argonaute (mRNA is cleaved)

If siRNA/mRNA is mismatched

mRNA is not cleaved; instead, there is translational inhibition

CRISPR

Clustered regularly interspaced short palindromic repeats

CRISPR in bacterial function

Recognize and disarm invading viruses

CRISPR-Cas technology

Allows scientists to edit genes and manipulate gene expression

Spacer DNA following CRISPR sequences

Match DNA sequences found in bacteriophage genomes

Cas9 enzyme

Endonuclease that cuts both strands of DNA at a specific site

Single guide RNA (sgRNA)

Engineered RNA that forms a complex with Cas9

sgRNA Guiding Region

part of the CRISPR RNA that is complementary to the target region so Cas9 can cut

sgRNA Scaffold Region

transactivating RNA that forms a multi-hairpin loop structure that binds to the Cas9 protein

Protospacer adjacent motif (PAM)

Sequence downstream of the target sequence and is required for Cas9 function

Step 1 of Cas9 DNA Cleavage

Cas9 binds to sgRNA

Step 2 of Cas9 DNA Cleavage

Cas9-sgRNA complex binds to a PAM site on the target DNA

Step 3 of Cas9 DNA Cleavage

Guiding region of the sgRNA binds to the target DNA sequence

Step 4 of Cas9 DNA Cleavage

Cas9 makes a double-stranded break 3 base pairs upstream of PAM

Step 5 of Cas9 DNA Cleavage

The complex releases from the DNA

Non-homologous end joining

Enzymes reconnect the ends of the double-stranded break back together

Non-homologous end joining Cons

May randomly insert or delete one or more bases and cause mutations

Homology directed repair

Proteins patch the break using donor template DNA including the desired sequence

Plasmid

Extrachromosomal DNA that is most common in bacteria, but sometimes in archaea and eukaryotes

Restriction enzyme

An enzyme that cleaves foreign DNA into fragments

Restriction enzymes are what group of enzymes

Endonuclease

Restriction enzymes mechanism

Two incisions through each backbone of the double helix

Why restriction enzymes dont cut your DNA

Methylation

Multiple cloning site (Polylinker)

Short DNA with many restriction sites to allow a piece of DNA to be inserted

Mirror-like Palindrome

Sequence reads the same forward and backward on a single strand of DNA

Inverted Repeat Palindrome (more common)

Sequence reads the same forward and backward on complementary strands

EcoRI digestion

Produces uneven, staggered, stable sticky ends and is a part of the restriction modification system

Smal restriction enzyme

Cleave produces blunt ends

Ligation

The T4 DNA ligase enzymes covalently connects the sugar backbone of two DNA fragments

First step of DNA Ligation Reaction

Sticky DNA ends collide by chance and stay together long enough for ligase to join them

Second step of DNA Ligation Reaction

Ligase catalyzes the joining of the 3’ OH to the 5’ phosphate

Insertion of a DNA fragment into a plasmid vector

Preferrably uses two dif restriction enzymes so sticky ends are produced

Blunt-ended PCR products

Less efficient and 100x slower than sticky end ligation

Standard Cloning insert to vector ratio

3 insert : 1 vector molar

Standard Insert/Vector DNA Ligation materials

Vector DNA, Insert DNA, ligase buffer, ligase, water

Blue/white screen

A technique that allows for the rapid detection of recombinant bacteria

White colonies represent

Cells transformed with vectors containing recombinant DNA

Blue colonies represent

Cells transformed with only the vector (nonrecombinant plasmids)