Gene Cloning and Expression in Bacteria

Why it it difficult/impossible to artificially synthesize proteins?

They are larger and more complex than small-molecule drugs

Give examples of therepeutic proteins

Hormones, blood factors, insulin, vaccines, antibodies

What are the two (three) methods for therapeutic design?

Rational protein time, directed evolution (and hybrid evolution)

What is meant by rational protein design?

Designing a protein with the correct folds to interact with a known target

Has become much easier with AI tools such as Alpha-Fold

What is meant by directed evolution (for protein design)?

Mutating genes and selecting for things that have greater effectiveness in a particular pathway

How did insulin used to be produced before the 1970s? What about after?

Before: human insulin obtained from cadavers

Now: recombinant protein obtained from bacteria containing human gene

What are the requirements for a successful host organism for a cloned gene?

• Easily cultured (in a bioreactor)

• Non-pathogenic

• Sufficiently well understood to allow genetic engineering for high expression of required proteins

• Ethical considerations

Give a named example of a cloned human gene for the treatment of an illness

Human growth hormone (required for growth and development) produced in pituitary gland

hGH purified from cadavers used in treatment of growth deficiencies until 1980s. Major HIV and prion transmission issues led to banning of natural hGH.

Now engineered in E. coli bacteria

Describe the structure of hGH

Simple molecule- single polypeptide with just two disulphide bonds

No cofactors required for activity

Non-toxic to host

Stable and soluble

Why is it important that the gene being cloned is non-toxic to its host?

Toxic protein is sequestered into large aggregates and loses its solubility

Describe the experiment where recombinant DNA was first made

Labs of Paul Berg, 1972

Took DNA from 2 viruses: SV40 virus and lambda phage

Both were cut of EcoRI and ligated together with T4 DNA ligase

Define recombinant DNA

DNA that has been formed artificially by combining constituents from different organisms

Describe clonal growth of bacteria

One bacterium produces two copies of itself and all bacterial DNA is copied

Individual clones amplify into c

What are the two sources of DNA in a bacterial cell?

Chromosomal DNA and plasmids (non-chromosomal DNA)

How do we know that antibiotics have existed for millions of years, being used by bacteria in their ‘warfare’ against other microbes?

There are antibiotic resistance-containing plasmids in bacteria discovered in millenia-old untouched ice sheets

Antibiotic resistance-containing plasmids are just something that bacteria have

How is bacteria’s inherent property of antibiotic resistance useful to molecular biologists?

It’s a way of knowing whether a bacterium has taken up a plasmid with the gene of interest on it

What are the three ways in which genes are horizontally transferred between bacteria

Transformation (see other flashcard)

Conjugation (“bacterial sex” where they make the tube and send a plasmid through it)

Transduction (phage infection)

Describe the process of bacterial gene transformation

A stress response in which bacteria take up DNA from the outside world on the off chance they take up the material which will save their lives

To exploit this property scientists give them heat shocks or put them in high-salt solutions to make them take up genes of interest

Describe the first gene cloning experiment

Uni of California 1973

Inserted frog (xenopus) DNA into bacterial plasmids to create the first transgenic organism

Digested both sets of DNA with EcoR1, combined and ligated, transferred back into bacteria and analysed clones of bacteria for frog DNA

Which parts of the bacterial plasmid must be left intact for a successful gene cloning experiment?

Antibiotic resistance region, origin of replication

What is used to stop the two ends of a bacterial plasmid from closing on itself?

Alkaline phosphatase removes a phosphate group and stops the ring closing on itself

What are the properties of a modern (engineered) plasmid? What problems to they solve?

A multiple cloning site (solves the problem of the plasmid being cut multiple times in multiple places)

LacZ gene (solves the problem of being able to tell which plasmids have undergone a successful gene transfer)

Origin of replication

What is the multiple cloning site?

A site with many restriction enzyme sites in it, that are only found there and nowhere else in the vector

It is in this region that the foreign DNA is inserted into, regardless of what the ends of the sequence happens to be

How does the Lac Z gene in an engineered plasmid help identify which plasmids have undergone successful gene transfer?

Lac Z gene encodes the alpha peptide (portion of beta galactosidase)

If cells contain the alpha peptide this is called alpha complementation

If cells are given a substrate called X-Gal, alpha peptide-containing cells have a blue colour precipitate in them

The inserted DNA disrupts the lac Z gene and means no blue!

What is meant by ‘compatible cohesive ends’?

Complimentary overhangs formed by digestion with DIFFERENT enzymes

Describe the process of introduction of restriction enzyme sites into PCR products

1:09:35

What is TA cloning?

Many Taq polymerases used in PCR leave a 3’ A-overhang and we can use this to clone

Basic cloning involves ligation of PCR into a product that has T overhangs

Not very efficient because only one base to pair

How is the efficiency of TA cloning improved?

Use topoisomerase to ligate DNA fragments instead of DNA ligase

The cloning vector is engineered to have the correct 5-bp recognition sequence for the enzyme to work

It covalently recombines DNA which is a fast reaction taking 5 mins at room temp

Gene of interest must still have an A overhang

How are bacterial cells ‘persuaded’ to express a protein of interest?

Add genes of interest under promoter sequences that are dependent on adding a sugar (eg. arabinose)

Why are genes inserted under the control of a promoter rather than constantly producing protein?

Inducible system is used because otherwise bacteria quickly gain mutations which mean they don’t have to expend energy producing a protein which does not benefit them

Why is mRNA used as a starting template for gene cloning rather than DNA?

Bacteria does not have RNA splicing

A cDNA copy made from RNA will not have introns

Why do some organisms show different preferences for the codons that they use to represent a particular amino acids?

Hypothesised to be matched to the abundance of tRNAs in cells

Why is it a problem for genetically engineered protein that different organisms have different codon preferences?

Need to re-engineer code to match the preferences of the host organism

Do this by creating synthetic genes

What are inclusion bodies?

Insoluble protein aggregates found in bacteria

Describe how the principles of the lac operon are used in cloning vectors?

Have a gene on plasmid encoding lac repressor

Stops T7 RNA polymerase from being made (T7 RNA polymerase is required for gene expression)

A chemical called IPTG is added, binding and sequestering the lac repressor

The lac repressor is moved so the T7 RNA polymerase gets made, which binds to gene of interest and expresses it

How are proteins ‘tagged’ for identifiaction and purification after cloning?

Just 3’ of the gene there is a sequence that encodes a tag

There is no stop codon so what is made is the protein of interest with a tag on the end

The tag is something that will adhere to a bead so it can be eluted later

What are some tags used for identifying proteins of interest during purification? [3]

His-tag: six histadine residues → nickel binding

Maltose binding protein (MBP): increased solubility

Glutathione S-transferase (GST): improved separation

How are proteins of interest eluted from the beads

Use a protease that cuts between protein of interest and the tag site

What is the process of phage display?

Phages can be induced to make antibodies

We make so many that they make every possible variant of human antibody

We find the phage expressing the correct antibody by giving them the antigen: where the phage sticks to the antigen they are bound while others are eluted

Example of a directed evolution experiment

Briefly describe the process of gateway cloning

An entry clone and destination vector are recombined using recombinase to create an expression clone (desired product) and byproduct