MMG 2040 - chapter 7 Expression Vectors

Describe the necessary features of shuttle vectors

They need:

• the origin of replication, selectable marker, gene of interest

• promoters, transcription sequence, poly-a site, termination site

properties: origin for species 1 + selectable marker for species 1, origin for species 2 + selectable marker for species 2, mcs

Describe the induction/repression of the lacUV and tet promoter

lac promoter, laci repressor, IPTG inducer

lacuv is typically off until you add IPTG (the inducer)

This is useful for inducible expression in bacteria

Compare promoter systems

lacUV: This is useful for inducible expression in bacteria

• lac promoter, laci repressor, IPTG inducer

• lacuv is typically off until you add IPTG (the inducer)

• inducible

T7: This is useful for very high protein production in bacteria

• T7 promoter + T7 RNA polymerase (phage-derived)

• Very strong transcription —> ex. producing a product

• constitutive

Tet-On/Tet-Off: this is useful for tight control in mammalian systems

• Tetracycline promoter used to drive gene expression (unlike in bacteria)

• Doxycycline added → transcription ON

• Doxycycline added → transcription OFF

• inducible

Name 4 methods of getting vectors into mammalian cell

1) Lipid-mediated transfection

2) Calcium phosphate

3) Electroporation

4) Viral transduction

Compare/contrast transient transfections and stable transfections

transient: not integrated in genome but remains in nucleus, not passed onto progeny/genetic alteration is not permanent, does not require selection, high copy number of material results in high level of protein expression, generally not suitable for studies using inducible vectors

• both DNA/RNA vectors can be used

• harvested 24-96 hrs after transfection

stable:integrated into genome carried stably from gen. to gen./genetic alteration is permanent, requires selective screening for stable transfectants, single/low copy number of integrated

Define transfection and transduction relative to mammalian expression vectors

transfection: Introduction of vectors, without using viruses, into mammalian cells

Transduction: Introduction of vectors into mammalian cells using viral-mediated delivery

Design a basic mammalian expression strategy

Bacterial or mammalian system?
Transient or stable?
Inducible or constitutive?
Viral or non-viral delivery?

lacUV

The lacUV5 promoter is a mutated promoter from the Escherichia coli lac operon very similar to the classical lac promoter,

• it requires no additional activators and it drives high levels of gene expression

The lacUV5 promoter sequence conforms more closely to the consensus sequence recognized by bacterial sigma factors than the traditional lac promoter does

• Due to this, lacUV5 recruits RNA Polymerase more effectively

  • leads to higher transcription of target genes

• lacUV5 works independently of activator proteins or other cis-regulatory elements

• While no activators are required, lacUV5 promoter expression can be regulated by the LacI repressor and can be induced with IPTG, which is an effective inducer of protein expression

  • Due to this control, the lacUV5 promoter is commonly found on expression plasmids

  • is used when controllable but high levels of a product are desired

t7

T7 expression vectors (commonly pET vectors) enable high-level protein production in E. coli by using T7 RNA polymerase to specifically transcribe a target gene

• system is induced by IPTG, which triggers the expression of T7 RNA polymerase from the host genome

• recognizes a T7 promoter on the plasmid to drive massive target gene expression

• Vector Construction: The target gene is inserted into a plasmid (e.g., pET) downstream of a strong T7 promoter.

• Host Cell: The vector is introduced into special E. coli strains that have the gene for T7 RNA polymerase integrated into their chromosome.

• Induction: T7 RNA polymerase expression is controlled by an inducible promoter (typically lacUV5). The addition of IPTG relieves repression, producing T7 RNA polymerase.

• Transcription: The produced T7 RNA polymerase specifically transcribes the target gene, leading to high-level protein overexpression.

• Tight Regulation: To prevent basal expression (leakiness) of potentially toxic proteins, T7 lysozyme (a natural inhibitor) can be expressed, or the system can use Lac repressor binding

tet off/on

Tet-Off and Tet-On systems are inducible gene expression tools that use tetracycline or its derivative doxycycline (Dox) to control gene activity.

• both relying on a transactivator protein binding to a Tetracycline Response Element (TRE) promoter

Tet-Off turns expression off when Dox is added

• requires tTA (Tet-Off): Tetracycline-controlled Transactivator

• The tTA protein binds to the TRE promoter, activating transcription of the target gene

• Dox binds to the tTA protein, preventing it from binding to the TRE. Expression turns OFF

• Useful for long-term studies where expression is usually needed, then turned off

Tet-On turns expression on when Dox is added

• requires rtTA (Tet-On): Reverse Tetracycline-controlled Transactivator

• The rtTA protein cannot bind the TRE. Expression is OFF

• Dox binds to the rtTA protein, inducing a conformational change that allows it to bind the TRE. Expression turns ON

• Often preferred for rapid response to Dox induction