lecture 2- recombinant DNA technology

Proteins could be effective pharmaceuticals while they are _______ to make.

difficult

_We usually use_________ or ____________ cells to make them

microorganisms, cultured mammalian

To make a protein drug, we need

The gene that codes for the protein (we don't usually create it from scratch).✅ A vector (like a plasmid) to carry the gene.✅ A way to insert the gene into the vector.✅ A way to get the vector into cells (like bacteria or mammalian cells).✅ A method to select cells that successfully received the gene.✅ A way to control when the gene is turned on to produce the protein.✅ A process to extract and purify the protein from the cells.

Scientist use the online data base__________to identify gene sequence of a target protein

NCIB

- How do we get the gene?

o Isolate the mRNA from cells that naturally produce targeted protein

o Covert mRNA into cDNA

o Use PCR (polymerase chain reaction) to make copies of the DNA of the targeted protein

steps of PCR

denaturation, annealing, elongation

denaturation of PCR

the step of PCR when the DNA sample is heated to 94 to separate strands

Annealing (PCR)

when the temperature is lowered (68)to enable the DNA primers to attach to the template DNA.

elongation (PCR)

A heat-tolerant DNA polymerase (Taq) copies the strands

PCR works on both ________and _______cells

living and dead

DNA manipulation enzymes play a role in

synthesis, attachment, breaking

synthesis/copying of DNA uses

DNA polymerase

attachment of two or more DNA uses

DNA Ligase

Breaking of DNA uses

restriction Enzymes

Example of restriction enzyme

restriction endonuclease

restriction endonuclease

A bacterial enzyme that recognizes a specific DNA nucleotide sequence and that cuts the double helix at a specific site within the sequence.

EcoRI

a restriction enzyme that specifically cuts DNA with sequence GAATTC and creates sticky ends

what is a plasmid

small circular piece of dsDNA found in bacteria

We use plasmids as ________ to carry the gene into cells.

vectors

Features of a Good Plasmid Vector

1. place to put the gene (cloning site)

2. selection mechanism(antibitoic resistant gene)

3. replication origin(a signal)

4. promoter (stop or start transcription)

not all bacteria have plasmids T or F

True

Vectors can be used to transfer genes to ____________,_________,_________and __________ cells

mammalian, yeast, insects, bacterial

How We Insert Genes into Plasmids- in bacteria & yeast: The process is called

transformation

How We Insert Genes into Plasmids- In mammalian cells: It's called

transfection

We use _________________- to identify cells that successfully took up the plasmid.

antibiotic resistance genes

If the bacteria grown in the presence of an antibiotic (penicillin), has received the plasmid, it will_________, if it did not it will__________

survive and grow, die

affinity tags on proteins

to make purification easier

what is subcloning

the process of putting the gene of interest into a plasmid

hosts for recombinant proteins

Bacteria

yeast

mammalian cells

pros of bacteria as a host cell

Most popular, Fast growth, cheap, express high levels of protein

Pros of yeast cells as host

Good alternative to bacterial, Can modify proteins post translational, good for complex proteins(no insoluble proteins due to multi step processing)

pros of mammalian cells for hosts

Best for human proteins, full modifications post translational

cons of bacteria cells for hosts

No post-translational modifications, some proteins misfold, can be trapped din inclusion bodies (mostly inactive)

cons of yeast cells for hosts

Cell and medium may contain compounds that affect binding of affinity tags, which makes them harder to isolate

cons of mammalian cells for hosts

Low expression levels, slow growth, expensive

If a protein needs modifications (like sugar attachments), ___________cells are best

mammalian