Biotech Quiz 2 Cards

Plasmid

self-replicating, double-stranded, circular DNA
molecule that is maintained in bacteria as independent
extrachromosomal entity.

Vector

genetically engineered plasmids that contain: origin of replication, antibiotic resistance gene, MCS (protein vs. enzyme – plasmid vs. vector)

Five main types of vectors

fertility F-plasmids, resistance R-plasmids, virulence V-plasmids, degradative plasmids, and Col plasmids

Fertility (F) plasmids

Contain transfer genes that allow genes to be transferred from one bacteria to another through conjugation

Episomes —> F Plasmids

Plasmids that can be inserted into chromosomal DNA

F positive / F negative

Bacteria that have/don’t have F plasmid

Resistance Plasmids

Contain genes that help a bacterial cell defend against environmental factors like poisons or antibiotics

Some resistance plasmids transfer themselves through conjugation, resulting in…

a strain of bacteria that becomes resistant to antibiotics

Virulence Plasmids

When a virulence plasmid is inside a bacterium, it turns that bacterium into a pathogen —> can be easily spread and replicated among affected individuals

Degradative Plasmids

Helps the host bacterium digest compounds that are not commonly found in nature, using its own special enzymes

Are degradative plasmids conjugative?

Yes!

Colicins Plasmids

Contain genes that make bacteriocins, proteins that kill other bacteria and thus defend the host bacterium

Replication Origin

Specific DNA sequence that must be present in a plasmid for it to initiate DNA replication

When host cell enzymes bind to ORI, what happens?

Initiation of replication of the circular plasmid

In bacteria, what is the origin called?

oriC, a single site where replication initiates bidirectionally

Variability of ORI in same/different species

Highly conserved, vary significantly

Mutations in the origin sequence or defects in initiation proteins leads to

DNA replication failure, leading to cell cycle arrest or cell death

How can info about ORI be used in the pharmaceutical world?

Inhibit proteins like DnaA that bind to the bacterial origin to block replication initiation

How do many antiviral drugs work?

Drug disrupt viral origin recognition or replacement proteins, which prevents the mechanism by which they hijack host replication machinery.

Selectable Marker

Carried by the vector to allow the selection of positively transformed cells

Antibiotic Resistance

Often used as a marker

Multiple Cloning Site (MCS)

Contains many unique restriction sites, which avoid cutting elsewhere in the vector.

Termination Sequence

Signals the end of transcription, ensuring RNA polymerase stops synthesizing the RNA transcript at the correct location

Termination Sequence prevents read-through

Prevents transcriptional read-through, which could lead to the expression of unnecessary or unintended downstream sequences

Structure of Termination Sequence

Includes hairpin loop that stabilizes the RNA polymerase-DNA complex

Rho-dependent terminators

Require the Rho protein to terminate the transcription

Rho-independent terminators

Rely on a GC-rich region followed by a poly-U sequence to form a hairpin loop, causing RNA polymerase to dissociate

Efficiency of Termination

Strong termination sequence ensures efficient and accurate termination, improving the overall expression of the target gene

Placement of Termination

Located downstream of the gene of interest in the expression vector to ensure control

Terminator Impact on Gene Expression

Proper termination is critical to avoiding wasteful transcription and ensuring high yields of the desired protein or RNA product

Poly-A Sequence

Sequence in DNA (AAAAA) that signal the addition of the poly-A tail to the 3’ end of the mRNA transcript during processing

Poly-A tail purpose

Protects mRNA from degradation, enhancing its stability, facilitates its export from the nucleus to the cytoplasm

Is Poly-A tail involved in terminating transcription?

No

Promoter

DNA sequence that initiates transcription of a gene by recruiting RNA polymerase and other transcription machinery

Strong/weak promoters

Drive high/low levels of gene expression

Constitutive promoters

Always active, leading to constant gene expression

Regulated Promoter

Activated only under specific conditions

Promoter Elements

TATA box, -10 and -35 elements, Enhancers

TATA Box

common in eukaryotic promoters, helps position RNA polymerase

-10 and -35 elements

Found in prokaryotic promoters, essential for RNA polymerase binding

Enhancers

Additional sequences that can increase promoter activity, often located up/downstream of the promoter

Considerations for promoters

Expression level, inducibility, host compatibility, leakiness

Insulators

DNA sequences that block or restrict the interaction between enhancers, silencers, and promoters, ensuring proper gene regulation

How do insulators help maintain the independence of gene expression?

By preventing unwanted activation or repression of nearby genes

Enhancer blocking insulators

Prevent enhancers from activating promoters of neighboring genes

Barrier insulators

Protect genes from the spread of heterochromatin and silencing effects

MOA

Insulators bind specific proteins that create physical barriers or loops in the DNA, isolating regulatory elements

How does MOA disrupt the interaction between enhancers and promoters?

By forming chromatin boundaries

Insulators prevent leakage

Ensure that enhancers and silencers in the vector or host genome do not interfere with the expression of the transgene

Insulators exhibit positional independence

Allow consistent expression of the transgene regardless of its integration site in the host genome

Insulators reduce variability

Minimize position effects, which can cause unpredictable expression levels

Retroviral and Lentiviral Vectors

Insulators are used to prevent silencing of the transgene due to integration into heterochromatic regions

Transgenic animals

Insulators help ensure consistent expression of the transgene across different tissues and developmental stages

pBR322 vector

Widely used plasmid that contains two antibiotic resistance genes. Used as an E. coli cloning vector

pUC vector

Series of plasmids derived from pBR322, which has a high copy number, ampicillin resistance gene, has multiple cloning sites (allowing for easy insertion of DNA fragments)

High Copy Number

Increases yield of plasmid DNA

pUC Vector useful for colony selection

Can test for antibiotic resistance and blue-white colony screening due to LacZalpha gene

pcDNA Vectors

Series of mammalian expression plasmids designed for high-level gene expression in mammalian cells

pcDNA Vector has CMV promoter and MCS

Strong immediate-early promoter, drives high level expression of inserted gene; allows for easy insertion of many DNA fragments

pcDNA expression vector system colony selection

Ampicillin resistance gene, has neomycin resistance gene which allows for selection using G418

Lac Operon

Set of genes in E. coli involved in the metabolism of lactose; classic example of gene regulation

Lac operon is only expressed when?

When lactose is present and glucose is absent, ensuring efficient energy use

Lac Repressor

Acts as a lactose sensor, normally binds to the operator region of the operon, blocking transcription

When present, lactose binds to the repressor, causing a conformational change that prevents what?

the repressor from binding to the operator

Catabolite Activator Protein (CAP)

Acts as a glucose sensor, binds to a site near the promoter and enhances transcription of the operon; only active when glucose levels are low —> cAMP levels are high

Mechanism of CAP

Glucose is low, cAMP levels are high, binds to CAP. cAMP-CAP complex binds to promoter region, enhancing RNA polymerase binding and increasing transcription

Why is lac Operon important?

Serves as a model for understanding gene regulation in prokaryotes, how cells adapt to their environment, important for molecular cloning and protein expression systems

IPTG

Chemical reagent mimicking allolactose, which removes a repressor from the lac operon to induce gene expression

Allolactose

Isomer of lactose, formed when lactose enters cells. Acts as an inducer to initiate transcription of genes in the lac operon. Encode proteins important for breaking down lactose

Metabolism of lactose occurs when?

When glucose is absent and lactose is present at high levels. To replenish the energy source, cells naturally break down lactose.

Why are antibiotic resistance genes important?

Helpful for selecting specific colonies to grow/develop

Bacteriophage

Virus that infects bacteria

Cosmid

a type of hybrid plasmid, a cloning vector used in genetic engineering, that contains the cos (cohesive end) site of bacteriophage lambda

Major distinction b/w Prokaryotic and Eukaryotic Cells

Euk cells have “true” nucleus containing DNA, Prok cells do not have a nucleus

Organelles present in Eukaryotes and absent in Prokaryotes

Nucleus!, Lysosomes, Endoplasmic Reticulum, Mitochondria

What factors affect choice of expression system?

(1) nature of target protein (2) solubility (3) functionality (4) yield requirements (5) intended use application (6) Generation Time

What is the most preferred system for protein expression?

E. coli!

What is the most preferred Eukaryotic protein expression system?

Insect cell lines

Three main insect expression systems

Baculovirus-insect cell system (BEVS), InsectSelect (IS) system, Drosophila expression system (DES)

Baculovirus

Rod-shaped viruses which have a large, circular, double stranded DNA genome.

Baculoviruses exclusively infect what?

Arthropods (mainly insects)

In baculovirus insect cell system, what is the most commonly used baculovirus?

AcNPV and BmNPV

During the infection AcNPV, which two viral proteins are synthesized by infected cells?

polyhedron and p10

Are polyhedron and p10 essential for the replication of the virus?

No!

How strong are the promoters of polyhedron and p10?

Remarkably strong!

Pros of Baculovirus expression system

increased solubility, able to perform complex PTM’s, high protein expression levels, lower cost, easy to scale up to bio reactors

What does it mean that baculoviruses are safe vectors?

They’re essentially nonpathogenic to mammals and plants

Drawbacks of Baculovirus expression system

restricted host range (specific invertebrate species)

What is the major advantage of yeast cultures?

They can be grown to very high densities

What are two most common yeast strains used in expression systems?

S. cerevisiae and P. pastoris

Benefits of S cerevisiae

Well described genetics and physiology, easily grown in small and large scale, strong promoters isolated/characterized, PTM’s, easily purified, GRAS by FDA

Application of Insect Engineering

Transgenic mosquitoes used to combat zika infection (males made sterile —> no babies)

Application of Yeast Engineering

Some American wines are being made with genetically modified wine yeast (lacking the gene that gives wine drinkers headaches)

YAC (Yeast Artificial Chromosomes)

engineered DNA molecules, used as cloning vectors in yeast cells, capable of carrying large DNA inserts for genome mapping and analysis. Mimic normal chromosomes.

YEP (Yeast Episomal Plasmid)

a type of plasmid, a small, extrachromosomal DNA molecule, that replicates independently of the yeast chromosome, allowing for high copy numbers

What is the order of operations for Yeast Cloning?

First transform E. coli, then introduce into yeast

Primary method of Yeast selection?

Use genes like HIS4 and LEU2 and media which does or does not contain His and Leu to select for Yeast with/without the genes.

Limitations of S cerevisiae

Hyper-glocosylation, purification issues (proteins in periplasmic space), ethanol toxicity at high cell densities

Benefits of P Pastoris

Genes regulated by methanol —> tight control; high cell densities with no risk of toxicity due to no ethanol, few proteins secreted (easy purification), high chromosomal integration