Synthetic Biology

Molecular Cloning

Lecture given by: Dilan B. Jaunky

Wednesday February 15th 2017

 Lecture outline
Review and Definitions

Cloning using Restriction Endonucleases

Cloning using BioBricks parts

The gateway system

Cloning without Restriction Endonucleases

o Gene splicing by overlap extension
o Cloning using in-vitro homologous recombination
o Cloning using in-vivo homologous recombination
 Review and Definitions
Gene Cloning: To place a foreign gene into bacterial cell for
further uses.

Restriction Endonucleases: An enzyme that recognizes

specific base sequences in DNA and cuts at or near those
sites (Recognizes palindromic sequences)

Recombinant DNA: The product of recombination between

two (or more) fragments of DNA. Can occur naturally in a
cell, or be fashioned by molecular biologists in vitro.

Vector: A DNA (a plasmid or a phage DNA) that serves as a

carrier in gene cloning experiments
 Restriction Endonuclease

https://www.neb.com/tools-and-resources/selection-charts/alphabetized-list-of-recognition-specificities

Recognize and cut

specific DNA
sequences
AsuII
e.g. AsuII cuts after
the first T in the
sequence TTCGAA

http://en.wikipedia.org/wiki/DNA_
ligase
 Restriction Endonuclease
DNA ligase: Can link together two DNA strands that
have double-strand break (a break in both
complementary strands of DNA)

DNA ligase

5
http://en.wikipedia.org/wiki/DNA_
ligase
 Molecular cloning
(plasmid)
pUC19 plasmid

The MCS is where

you will insert your
gene of interest
 Molecular cloning
EcoR1 EcoR1
EcoR1 cut site

EcoR1 EcoR1 EcoR1

EcoR1
EcoR1
RE

1) Cut plasmid DNA with EcoR1 Restriction Enzyme

2) De-phosphorylate the cut vector (alkaline phosphatase)
3) Mix de-phosphorylated vector and insert + DNA ligase and ATP
4) Obtain new vector with gene of interest
5) Transform into host cell and select

*De-phosphorylation of cut vector will reduce the number of false positive

Molecular cloning -
Directionality EcoR1 BamH1
EcoR1 cut site
BamH1 BamH1
EcoR1 BamH1
EcoR1 EcoR1
BamH1
RE

1) Cut plasmid DNA with EcoR1 and BamH1 restriction enzymes

2) Mix cut vector and insert + DNA ligase and ATP
3) Obtain insert with a specific orientation
4) Transform into host cell and select for transformant

*Digestion of the vector with two restriction endonucleases prevents re-

ligation
BioBricks cloning
strategy

Spe1= ACTAGT
Xba1=TCTAGA
Cloning without RE
strategies
How can we simplify and make it amendable
to easy
cassette construction?

Use:
Assembly using overlap extension
In-vitro homologous recombination
(Gibson Assembly)
In-vivo homologous recombination
(Yeast Homologous Recombination)
Gene splicing by overlap Extension -
SOEing
Gene 1 Gene 2

PCR PCR
Requirements:
- Primers designed with
homologous region
- PCR amplification and
isolation
- Incubation of both
Mix, denature and anneal
constructs with end
primers
- PCR reaction
Extend
- Isolation of full
construct

http://www.biotechniques.com/BiotechniquesJournal/2013/March/Gene-Splicing-by-Overlap-
Extension-Tailor-Made-Genes-Using-the-Polymerase-Chain-Reaction/biotechniques-341027.html
 Gibson Assembly

From: neb.ca

- Gibson assembly master mix is costly

- Regions of homology needs to be well
Gibson Assembly Multiple
fragments

From neb.com

Higher amount of parts in the Gibson assembly

reaction decreases the efficiency.
 Gibson Assembly

From genengnews.com
In vivo assembly Yeast homologous
recombination