University of Indonesia / Jakarta

March 1st 2019

Genetic Engineering: State of the Art and Trends

Hans-Jürgen Mägert, Anhalt University of Applied Sciences, Köthen, Germany

Genetic Engineering: State of the Art and Trends

1. INTRODUCTION

Definition of genetic engineering, milestones in history

2. MAIN PART

2.1. Classical genetic engineering

– Recombination by restriction and ligation
– Generation of libraries
– DNA/DNA hybridization

2.2. Advanced classical genetic engineering

– Polymerase chain reaction (PCR)
– T-vector cloning of amplicons
– Recombination by use of recombinases
– Recombination by PCR

2.3. New era in genetic engineering / trends

– Next generation sequencing
– In silico screening for functional genes / proteins
– Synthetic biology
– Using DNA as memory
– CRISPR/Cas9 system for genome editing
 Synthetic Biology

– The re-design and fabrication of existing biological systems

– The design and fabrication of biological components and systems

that do not already exist in the natural world

– More extensive and complex change of an organism compared to

classical genetic engineering – even generation of a novel organism
possible

– Availability of libraries with well-defined genetic building blocks

(„biobricks“)

– Use of these building blocks for generation of new organisms

producing or doing whatever you want
 DNA Synthesizer

Synthesized
genetic
building blocks

Nucleus

Cell Mitochondria

Products
http://www.synbioproject.org
 Applications

– Metabolic engineering (including generation of novel metabolic

pathways) - production of desired chemical compounds

– Detection of chemical pollutants and weapons

– Bioremediation

– Disease diagnosis and treatment

– Generation of biofuel

– Much more.....
 Advantages Compared to Classical Genetic
Engineering

– Already existing, well-defined, in parts compatible „biobricks“

– Use of optimized genes/cDNAs (e.g. concerning codon usage

or an optimized product)

– Use of ideal promoter regions (e.g. concerning activity and

induction - by certain compounds, heat, light etc.....)

– Possibility to generate an organism with ideal characteristics

for a specific application
http://parts.igem.org/Main_Page
http://parts.igem.org/Main_Page
http://partsregistry.org/
 There are five ways to get this part

1. You can find it in one of the Registry distributions

2. You can request it from the Registry

3. You can use PCR to extract it from a natural DNA sample

4. You can order it from a DNA synthesis company

5. Or, for short parts, you can assemble it from oligos

 There are five ways to get this part

1. You can find it in one of the Registry distributions

2. You can request it from the Registry

3. You can use PCR to extract it from a natural DNA sample

4. You can order it from a DNA synthesis company

5. Or, for short parts, you can assemble it from oligos

http://www.blueheronbio.com/
Fusion of „biobricks“ in the correct order
Fusion PCR (with restrictions), „Gibson
Assembly “ , and „Golden Gate
Cloning“ enable rapid directed assembly of
a large number of DNA fragments
=> required for complex genetic changes in
„synthetic biology“
Principle of „Gibson Assembly“
Principle of „Golden Gate Cloning“
 Principle of „Golden Gate Cloning“

– The restriction enzyme Bsa I recognizes a hexanucleotide sequence

but cleaves outside of this sequence generating a four nucleotide overhang

– One may design PCR-primers containing the Bsa I recognition site and a cleavage
site with a defined sequence (256 possibilities, e.g. for the reverse primer of the first
fragment) within a 5‘-“add on“ – sequence

– These sites may also be included in one of the primers for the next fragment to be
connected (here the forward primer for the second fragment)

– Fragments are generated by PCR using the primers with the add on – sequences

– Each fragment which has to be connected contains a specific cleavage site, the
generated overhang of which (sticky end) exactly matches the overhang of the next
fragment

– This enables a correct directed ligation of the fragments after cleavage by Bsa I

– Primers may be designed in that way, that the Bsa I – site is removed by the
cleavage
Nowadays desired DNA fragments may be ordered from companies for low prices
Craig Venter Institute generated a minimal
bacterial genome as a basis for creation of
novel organisms fulfilling any desired
function
https://www.igem.org/
Source: https://www.igem.org/
Source: https://www.igem.org/
Source: http://makezine.com/2013/05/16/diy-synthetic-biology-making-your-own-glowing-plants/
www.echromi.com
CRISPR/Cas9

By Emmanuelle Charpentier and Jennifer Doudna

The novel CRISPR/Cas9 technology enables highly

specific (targeted) manipulations of the genome

CRISPR = Clustered Regularly Interspaced Short

Palindromic Repeats

–> Lecture „Special Topics in Cell Biology and

Cell Culture“
 Using DNA as Memory

– The group of Nick Goldman and Ewan Birney encoded computer files totalling 739
kilobytes of hard disk storage (including Martin Luther King‘s „I have a dream
speach“) into a DNA code and synthesized it

– By DNA sequencing the original files were reconstructed with 100% accuracy

– Potential for cost-effective long time storage of informations

– 100 million hours of high resolution video data would fit into a small teacup

– No current required for storage

– Current cost approximately 12,400 Dollars for storage and 220 Dollars for decoding
per megabyte –> shall decrease significantly within the next years

– Goldman et al.: Towards practical, high capacity, low-maintenance information

storage in sythesized DNA
Nature 2013 (Epub ahead of print)
Ting Cui Gao Xu Anna Feulner
SLS
Moffel
Caution! – This is a Joke!
Thank you very much for your attention!