DNA Cloning/Gene Cloning/Molecular Cloning

Cloning

way to let E.coli store and replicate DNA sequence in useful form

use of cloning

• produce large quantities of protein in E.coli or other easy-to-grow organism

• Make recombinant DNA molecules in use in organism of choice: What is effect of a particular mutation in a gene? What is effect of changing expression level of a gene? Express fluorescent protein in specific cell type? Genome editing through CRISPR

• E.coli allows for selection of correctly assembles piece of DNA and copies DNA more cheap and accurate than PCR

Recombinant DNA

cloning DNA from different source s

Plasmid

cloning vector

Gene of interest has

origin of replication and antibiotic/selectable resistance marker

Put plasmid into bacterium by transformation

putting DNA into bacteria plate on media containing antibiotic (select cells containing plasmid)

Allow bacterium to reproduce

propagate plasmid, extra plasmid put into another organism

Clone of cells

E.coli cells

1st step of cloning

amplify a gene/sequence of interest by PCR

2nd step of cloning

place PCR production into a plasmid (cloning vector)

3rd step of cloning

transform your gene and vector into E.coli

4th step of cloning

select for E.coli that have drug resistance (conferred by the plasmid)

5th step of cloning

Confirm that the new plasmid sequence is correct

6th step of cloning

grow lots of E.coli to produce more plasmid (or protein)

Gibson assembly

common cloning method (closely related methods with different names), versatile

Key features of Gibson assembly

• connect pieces of DNA together with exact junctions of choosing

• can assemble >2 pieces simultaneously

• must 1st add homologous sequences to molecule that you want to connect

can add any sequence to 5’ ends of PCR primers

Designing primers for Gibson Assembly requires 2 parts

1. template specific portion: PCR primers for insert

2. Vector specific tail: overlap, homology arms of lineralized plasmids

How Gibson Assembly connects DNA fragments together

3 key enzymes, enzyme mix

exonuclease, DNA polymerase, DNA ligase

Exonuclease

removes some of the nucleotides from the 5’ ends of both the insert and the backbone (linearized section of plasmid)

DNA polymerase

fills in the missing nucleotides but there are still breaks in the phosphodiester backbone

DNA ligase

seals the nicks by forming new phosphodiester bonds, leaving a fully circular double-stranded molecule

Nicks

breaks in the phosphodiester backbone