Fundamental Bioscience CHEM-C1300

Lecture 11a Biotechnology (Ch. 20):

• Molecular cloning
• Production of proteins Recombinant
• Dideoxy sequencing plasmid

Lecture 11b Viruses (Ch. 33): Slides modified from Pearson

• Different viruses
• Replication of corona virus
• Reverse transcriptase

Silvan Scheller
Assistant Professor for Biochemistry
silvan.scheller@aalto.fi (Ke1 D328a)
Image: © Andriy Onufriyenko/Getty Images

March 29th 2023

 Content
Chapters 4 + 15-20 + 33
• 4: What is DNA, RNA?
• 15: How is DNA replicated and repaired? /PCR
• 16: How do genes work?
• The central dogma in Molecular Biology: Information flow goes from
DNA à mRNA à protein à metabolism / reactions / “action”
• 17: How does this work mechanistically?
• 18/19: How is this flow of information controlled?
• 20: How can we use these processes for biotechnology?
• 33: How do viruses work? e.g. corona-virus is an RNA virus
 Learning Outcomes
Chapter 20
Be able to explain:
• how recombinant DNA technology works
• methods to analyze mRNA
• how dideoxy sequencing works

Viruses (Ch. 33)

Be able to describe:
• different classes of viruses based on their genomes
• how corona viruses replicate
• exceptions of the “central dogma in molecular biology”
 Application of Central Dogma
Protein production

DNA

Introduce
mRNA
(synthetic) DNA

mRNA

“Harvest” Protein
(customized) Proteins
 Plasmid + lac Promotor

à The easiest tool for Ribosomes

genetic modifications

à Can be used as a Plasmid

cloning vector Cytoplasm

Chromosome
à Use lac promotor to
Plasma 1 µm
control the expression membrane
Cell wall

lac operon

DNA Regulatory
lacI lacI sequence Promoter Operator lacZ lacY lacA
promoter of lac operon
 Genetic Engineering - Recombinant DNA

Gene 1. Identify a palindromic

Recognition site recognition site. Attach
Recognition sites
(palindrome) same recognition site to
(same palindrome
cDNA gene.
as in plasmid)
Plasmid

Restriction
endonuclease
(EcoRI) 2. Add restriction
endonuclease.
 Inserting Genes into Plasmids

3. Sticky ends result.

Sticky ends Sticky ends

Gene

4. Insert gene into

Recombinant plasmid.
plasmid
 Transformation

Recombinant
plasmid

Transformation
 Recombinant Protein Expression
Protein expression vector (plasmid)

• Origin of replication
allows multiple
copies to be
produced in the cell
• The selection
marker is typically
an antibiotic
resistance gene
• Gene encoding the
protein of interest
 Plasmids and Typical Components

https://www.snapgene.com
 Protein Expression Vector
Producing His-tagged proteins:
• 6–10 histidine codons (the “His-tag”)
are cloned upstream or downstream of
the target gene
• The His-tag cause the protein to
adhere to a column containing
immobilized dicationic metals such as
Zn2+ or Ni2+ (i.e., immobilized metal
affinity chromatography or IMAC)
 Applied Example for Molecular Cloning:
How to get Enzymes from Uncultured Deep-sea microbes?

e.g. propane-oxidizing
enzyme within this microbe

CO2 H2S

e-

5 µm
alkane SO42-

Archaea Bacteria Some microbes convert

Depth= 874 m, Temp= 4.02 °C propane to CO2

Problems:
• Microbes have doubling time of months
• Very low amount of biomass in sample à What to do?
 Applied Example for Molecular Cloning:
How can I study an enzyme from an uncultured microbe?
1) Get sample (100mg)

6) PCR

13) Purify the protein (e.g. with Ni-column)

14) Make experiments
 Column Chromatography (in General)

A mixture of (cellular) proteins is separated as a result of differential interactions with

the column matrix.
The more a protein interacts with the matrix, the later it will elute from the column
 Affinity Chromatography (e.g. Ni2+)
Purification of Proteins with His6-tag

His-tagged target
protein

eluted with an
imidazole buffer
Possible Application for Metabolic Engineering:
Current process with wild-type (=“natural”) archaea:

CO2
+ METHANE Renewable Feed into existing
CH4 methane gas grid
4 H2 + 2 H2O
Electrolysis
microbial

e-METHANE
Possible Application for Metabolic Engineering:
Current process with wild-type (=“natural”) archaea:

CO2
+ METHANE Renewable Feed into existing
CH4 methane gas grid
4 H2 + 2 H2O
Electrolysis
microbial

e-METHANE

Exchange with DNA from propane-oxidizing microbe

à could it be possible to produce propane from CO2? à To be tested…
Storage
Drop-in fuel
Renewable ship fuel
3 CO2 (LPG)
+ PROPANE Electricity
C3H8
10 H2 + 6 H2 O
Electrolysis Polypropylene &
microbial Propylene C3 chemicals
e-PROPANE Renewable jet fuel

à See next week how to exchange genes in the chromosome

How to Determine the Exact DNA Sequence?

Remember PCR: Polymerase Chain Reaction

Taq polymerase

1. Create reaction
mixture.

dNTPs Primers
 Dideoxy Sequencing: Base Sequence of DNA

Dideoxy Adenine Triphosphate ddATP

 Dideoxy Sequencing

Normal dNTPs ddNTPs

(extend DNA strand) (terminate synthesis)
No OH

1. Incubate
reaction mixture.
Each ddNTP
has a different
fluorescent label
 Dideoxy Sequencing
Template DNA

2. DNA synthesis
occurs.

Primer Non-template DNA

3. Collect DNA
strands that are
produced.
 Capillary Electrophoresis

Long fragments Short fragments

4. Separate
fragments.

Output

5. Read output.

Sequence of non-template DNA

 Introduction to Viruses

• A virus is an obligate, intracellular parasite

• Viruses enter a host cell and use the host’s biosynthetic machinery to
reproduce and synthesize its proteins
• Most biologists would argue that viruses are not alive, because they
depend on their host cell to satisfy the key attributes of life
• Viruses
– Have a genome
– Are well adapted
– Evolve
• Nearly all organisms examined thus far are parasitized by at least one kind
of virus
 Comparison Viruses vs. Organisms

© 2017 Pearson Education, Ltd.

 Size
Eukaryotic cell
(human red blood cell)

7 µm
Bacterial cell
(E. coli)
2 µm

0.1 µm
Virus particles
(HIV)
© 2017 Pearson Education, Ltd.
 Analyzing Morphological Traits

• In terms of overall structure, most viruses fall into just two

general categories:
1. Those enclosed only by a protein shell called a capsid
2. Those enclosed by both a capsid and one or more
membranous envelopes
 Different Morphologies
(a) Tobacco mosaic virus (b) Adenovirus (c) Bacteriophage T4 (d) Smallpox virus

50 nm 100 nm 50 nm 100 nm

Genome Genome Genome Genome

Protein capsid Protein capsid Protein capsid

Protein
capsid

Membranous envelopes
© 2017 Pearson Education, Ltd.
 How Do Viruses Produce Proteins?
• Viral mRNAs that code for envelope proteins are
– Translated as if they were mRNAs for the cell’s own
membrane proteins
• mRNAs that code for capsid proteins are
– Translated by free cytosolic ribosomes as if they were
cellular mRNAs for cytosolic proteins

• In some viruses, long polypeptides called polyproteins are cut

into functional proteins by a viral protease (e.g. corona
viruses)
 How Do Viruses Copy Their Genomes?

• Viruses with an RNA genome must supply their own enzyme

to make copies of their genome
• Most RNA viruses use an RNA polymerase called RNA
replicase, which synthesizes RNA from an RNA template
– Synthesis uses ribonucleotides provided by the
host cell
 Viruses with RNA Genome
(+)ssRNA
Genomic strand

Replicase

dsRNA intermediate

Template strand

Replicase

© 2017 Pearson Education, Ltd.

(+)ssRNA products (genome copies)
 Overview of Different Genome Types (fig 33.17)

DNA viruses RNA viruses Reverse-transcribing viruses

Class I Class II Class III Class IV Class V Class VI Class VII

dsDNA ssDNA dsRNA (+)ssRNA (–)ssRNA (+)ssRNA (RT) dsDNA (RT)

Reverse
transcription

Reverse
transcription

mRNA

To viral mRNA and proteins

Proteins
Genome replication

© 2017 Pearson Education, Ltd.

 Positive-Sense Single-Stranded RNA
([+]ssRNA) Viruses

• Most of the commercially important plant viruses belong to

this group
• Members of this group also
– Parasitize bacteria, fungi, and animals
– Include the viruses that cause the common cold, polio,
SARS-CoV, West Nile virus, and hepatitis in humans
 Coronaviruses have (+)RNA Genomes

2019-CoV has 79% nucleotide identity to SARS-CoV

This is (+)RNA

Wit et al. Nat. Rev. Microbiol. 2016, 523

Overall Replication

(-)RNA

Subgenomic (+)mRNA

Wit et al. Nat. Rev. Microbiol. 2016, 523

 Test for Corona Virus

• Most reliable detection method is to confirm presence of the

virus’ genome (or part of it)

• Genome needs to be known

• Use of PCR

• RNA genome needs to be first converted to DNA

àusing reverse transcriptase (see virus class VI)
 Retroviruses
• Some RNA viruses, such as HIV, are retroviruses
– In retroviruses, the RNA genome is transcribed to DNA by
a viral enzyme called reverse transcriptase
• Reverse transcriptase is a DNA polymerase that
– Makes a single-stranded complementary DNA (cDNA)
from a single-stranded RNA template
– Removes the RNA strand and synthesizes the
complementary DNA strand, resulting in double-stranded
DNA
• The cDNA copy of the genome is then inserted by a viral
protein into the host-cell chromosome
 Reverse Transcriptase

cDNA Double-stranded cDNA

cDNA
template
RNA template

First, reverse transcriptase Then, reverse transcriptase

synthesizes cDNA from RNA synthesizes double-stranded
DNA from cDNA

© 2017 Pearson Education, Ltd.

 Antiretroviral Therapies
Inhibiting the reverse transcriptase (RT) of HIV:

Didanosin (not in use anymore) Zidovudine = azidothymidine

These drugs are phosphorylated in vivo to their triphosphates, which inhibit RT

2nd effect if they get incorporated:
Terminate DNA synthesis
à see: ddNTPs for dideoxy sequencing
 Tasks:
Biotechnology (Ch. 20)
• Study Ch. 20.1 – 20.2 carefully
• Study “Bioskill 10”, p. 80 – 84 (some parts will be discussed next week)
• Pearson Exercises: 11a (10pt, Tutorials); 11b (16pt, Quiz)

Viruses (Ch. 33)

• Read chapter 33: “big-picture” is part of the exam
• e.g. figure 33.17: classes of viruses
• Voluntary: read beginning of Wit et al. 2016 paper:
• p. 523-524 + first 21 lines on p. 525
• Figures 1+2

• Revise course