BMB 829

Special Problems in Macromolecular Analysis

and Synthesis

Erich Grotewold
[email protected]
Office in 210A Biochemistry Building
 BMB 829
Special Problems in Macromolecular Analysis and
Synthesis

Weeks 1 - 4 (3 hrs/week) Weeks 5 - 9 (3 hrs/week) Weeks 10 - 14 (3 hrs/week)

MODULE 2 MODULE 3
(829.001) (829.003)

Recombinant DNA & Molecular Interactions

MODULE 1 Genome Editing
(829.001)

Intro to Methods in
Biochemistry &
Molecular Biology

Separation & Analysis of Molecular Structure

Cells & Biomolecules Elucidation

MODULE 4 MODULE 5
(829.004) (829.005)
 Module 1
Class # Date Instructor Topic
1 Tuesday, August 29 Erich Grotewold Module introduction. Introduction to model organisms, macromolecules and their interactions,
microscopy, and recombinant DNA methods.

2 Thursday, August 31 Erich Grotewold Introduction to protein biochemistry.

Melinda Frame
Visit to the Center for Advanced Microscopy

3 Tuesday, September 5 Erich Grotewold Introduction to protein-protein, protein-nucleic acid and protein-small molecule interactions
Ben Orlando Introduction to protein structure analysis

4 Thursday, September Erich Grotewold Team Assignment 1

7

5 Tuesday, September Erich Grotewold Team Assignment 2

12

6 Thursday, September Kevin Childs Visit to the Genomics Core

14
Erika Lisabeth
Visit to the Drug Discovery Core

7 Tuesday, September Erich Grotewold Team Assignment 3

19

8 Thursday, September Erich Grotewold Team Assignment 4

21

9 Tuesday, September Tony Schilmiller Introduction to Mass Spectrometry – visit to the Mass Spectrometry Core
26
 Rules for the Class
• Hopefully, you are taking this class because you are interested
and think that you can use it for your PhD
• Masks are optional but mandatory for anybody feeling unwell
• Classes and facility visits are mandatory – if you have a
problem that prevents you from attending, provide a justification
prior to the class – no class can be missed without justification
• Grading for the module will be based on overall participation
(25%), team assignments discussed in class (40%), and weekly
homework assignments (35%) based on the topics already
covered in the module and aimed at assessing basic concepts
and understanding through problem-solving questions
 Team Assignment
(Full document on D2L)
The CDC just determined that a new pandemic is underway and decided to enlist the
help of BMB 829-301 students, known for their ability to solve complex problems. CDC
scientists were able to isolate enough of the infective materials to know that it is a
Coronavirus, although it is clearly not a variant of what caused COVID-19. The CDC
will provide BMB 829-301 students with enough quantity of the new virus (VirusX for
now, feel free to rename) to perform the research necessary for a comprehensive
response to this new pandemic. The research consists of four parts, each representing
a team assignment:

Assignment 1: Develop a PCR-based kit for the fast and accurate diagnosis of
VirusX

Assignment 2: Establish the molecular interactions between VirusX and host cellular
proteins

Assignment 3: Identify small molecules that serve as potential therapeutic drugs

Assignment 4: Develop a vaccine against VirusX

 Team Assignment – How will it work?
(Full document on D2L)
Class will be divided into teams of 2 – 4 students – discuss among yourself and give
me a list of the teams by Thursday. Each team will develop one specific aspect of the
larger problem provided and present it to the class at the appropriate date.

Presentations need to be prepared in PowerPoint and should consist of a brief

introduction to the problem, an explanation of the problem itself and what the
objectives to be accomplished are, a detailed description of the methods to be used
including enough technical information for everybody to understand the procedures
and how they will be applied. The presentation should end with a discussion of the
potential problems that could be encountered and how the team would expect to
overcome them. All team members should present and should be ready to answer
questions about any aspect of the presentation (in other words, the entire team needs
to be familiar with the entire project presented, no specialization of members with just
a particular aspect of the project). Presentations should be planned to take 40 – 60
min, and not more than one slide per minute is recommended (of course less slides
are OK, if the slides are very information rich). The grade for the assignment will be
based on the appropriateness and completeness of the project, the quality and clarity
of the slides, the way the slides are presented and the ability to address questions.
Each member of the team will receive a separate score.
 Homeworks
Each Tuesday I will provide the class a printed set of questions or problems to answer
or resolve and return to me as a hard (printed document) at the beginning of the
class the following Tuesday before the class starts. Failure to return will result in
getting zero points for that homework. No late submissions will be considered.

There will be a total of three homeworks, with the first one today

I can’t police how you do your homework; hence you can do them as a team and use
any source of information. However, the document I receive must be original (e.g.,
identical documents or sections will be nullified, and no copying/pasting from the web)

If time allows, we can discuss any problem/question at class once the homeworks
have been returned
 Today
Introduction to model organisms
Macromolecules and their interactions
Microscopy introduction
Recombinant DNA methods
 Model Organisms
A model organism is a species that is extensively studied to
understand particular biological phenomena, with the
expectation that discoveries made in the model organism will
provide insight into the workings of other organisms1
What makes a good model organism?
How has this changed over time?
Examples?
Limitations?

1
Fields & Johnston, 2005, Science 307: 1885-1886
 Molecular Interactions
Everything in biology is about molecular interactions:
Protein-protein
DNA-DNA, DNA-RNA, RNA-RNA
Protein-DNA
Protein-RNA
Protein-small molecule
DNA-small molecule
etc..
 Molecular Interactions
Very important that it is clear what is the difference between
covalent versus non-covalent interactions – please provide
examples
 Noncovalent Interactions
Three types of intermolecular forces (covalent are intramolecular by
definition of a molecule)
• Van der Waals forces – occur because of temporary or
permanent dipoles
• London dispersion forces occur due a temporary dipole
• Dipole-dipole interactions
• Ion-dipole interactions
• Hydrogen bondings
• Hydrophobic forces
Macromolecules, including proteins and DNA, are NEVER in
solution; they are in suspension – thus DNA is not dissolved,
it is resuspended – precipitation is actually aggregation
 Imaging Methods
Fluorescence microscopy

Electron microscopy Magnetic resonance imaging (MRI)

1 nm 10 nm 0.1 μm 1 μm 10 μm 0.1 mm 1 mm 1 cm 10 cm 1m

Optical microscopy

Confocal microscopy

A microscope cannot resolve two objects located closer than ~λ/2

 Introduction to recombinant DNA methods

Recombinant DNA technology involves using enzymes and

various techniques to isolate, manipulate and characterize DNA
segments of interest

Many different techniques and tools – we will have a

chance to cover only a few, but if there is any that you
would like to discuss in more detail, please let me know

Synthesis/isolation  Modification  Cloning  Analysis

I strongly recommend Molecular Cloning: A Laboratory Manual by Sambrook

(Cold Spring Harbor Labs Press) – three volumes covering pretty much any
molecular biology technique – never outdated; great resource for the lab
 DNA Synthesis: Chemical Synthesis
Will be the 3’ end of the oligo
Cost today is as low
as $0.07 per base for
25 – 50 nmoles and
there are hundred’s of
companies that will do
it

For PCR, usually

purification is needed
for lengths >100 bp –
Why?

Some jargon…
Oligo = oligonucleotide
(often called primer
when used for PCR)
Hughes et al, 2011 Methods in Enzymology 498: 277-309
 DNA Synthesis: From RNA
• DNA can be synthesized from RNA by the action of a reverse
transcriptase (RT)
• Many different types of RTs available – require a primer, can use oligo dT
or random short oligonucleotides – when one or the other? What is the
product of the synthesis?

Just in case that we forgot the Central Dogma of Biology

 DNA Synthesis: PCR

https://www.genome.gov/genetics-glossary/Polymerase-Chain-
Reaction

Yes, there was molecular biology before PCR (first used by Kary Mullis in 1983)…
 Nucleic Acids Isolation
• DNA can be extracted from pretty much anywhere – yields will vary
of course
• Consider what your sample contains, and what is the purpose of
the DNA that you plan to isolate (do you need 100 μg or are 10 ng
enough? Does it need to be rather pure?)
• DNA is rather stable at pH 6 – 8 and in the presence of chelating
agents (EDTA; 1 – 10 mM) helps – why?
• Before the advent of DNA purification columns (mostly silica-based,
but can also be ion exchange columns), selective precipitation of
nucleic acids with alcohols (ethanol, isopropanol) provided a
powerful tool –why does ethanol precipitate DNA and other nucleic
acids?
• RNA extraction is usually complicated by tissues having high
quantities of small, thermostable RNAses forcing to work fast, in the
cold and using chaotropic agents
 Plasmids
• Extrachromosomal, double stranded
circular DNA present in most bacteria
• Relaxed replication (as opposed to
stringent), meaning that it is
independent of the replication of the
bacterial genome
• Medium copy number (~20 per cell) –
can be increased with chloramphenicol
30-50 times
• Many modern plasmids (e.g., pUC)
derived from pBR322. Most carry
mutation in ori that increase number to
500 – 1,000 copies/cell
Note:
Origin of replication (ori)
Selectable markers (amp, tet)
Restriction sites for cloning
 The Magic of Plasmid Isolation
E. coli culture
Harvest by centrifugation and resuspend in
10 mM EDTA glucose, Tris

Bacterial suspension

Add lysis solution (NaOH, SDS) and mix

Lysate

Add KAcO, mix, incubate in ice for 10 min - centrifuge

Supernatant
(optional, you can do a
phenol-chloroform extraction – Add 2 volumes ethanol, incubate in ice and centrifuge
how does it work?)

Pellet is your plasmid DNA (from 3 ml of culture you get 5

– 10 μg plasmid DNA clean enough for most procedures)
 Nucleic Acid Manipulations
The toys of the molecular biologist
• DNA can be cut at specific sequences by restriction
enzymes
• DNA fragments can be pieced together using ligases
• Phosphate groups can be added or removed at the ends of
DNA by kinases and phosphatases, respectively
• DNA can be copied by polymerases (ALL work 5’  3’)
• RNA can be converted into DNA by reverse transcriptases
• DNA can be transcribed by RNA polymerases
• RNA can be translated by in vitro translation systems
 Restriction Enzymes
• Restriction enzymes are a group of endonucleases that cut
double-stranded DNA
• Type I recognize specific DNA sequences but make their cut
at seemingly random sites that can be as far as 1,000 base
pairs away from the recognition site
• Type II recognize and cut directly within the recognition site.
Recognition sites are generally 4 to 6 base pairs in length
and can be palindromes, i.e., a region of DNA in which the
sequence of nucleotides is identical with an inverted
sequence in the complementary strand
• Type III recognize specific sequences but make their cut at a
different specific location that is usually within ~25 bps of the
recognition site
 Type II Restriction Enzymes
• Restriction enzymes are derived from various bacteria or plasmids, hence the
names – e.g., EcoRI from Escherichia coli plasmid RI
• Restriction yields blunt ends or sticky ends. Sticky ends can only be ligated to
complementary/compatible ends, either cut with the same enzyme or with
enzymes that yield compatible ends

EcoRI site:


GAATTC

CTTAAG


EcoRV site:


GATATC

CTATAG

Why are bacteria making restriction
enzymes?

Why is there own DNA not digested?

 DNA Methylases

in vitro in vivo

host DNA protection

 A Few Other Facts
• Not all restriction enzymes care about DNA methylation
• Restriction enzymes can use different buffers and salt
concentration for idea activity
• Restriction enzymes sometimes display “star activity” in
suboptimal buffer conditions (e.g., different salt concentration)
• New England Labs has one of the best information on restriction
enzymes preferences – see
https://www.neb.com/tools-and-resources/usage-guidelines/neb
uffer-performance-chart-with-restriction-enzymes

Restriction enzymes are proteins, hence need to be

stored adequately – How? Why? Consequences?
 DNA Ligases
• Facilitate the joining of DNA strands by catalyzing the
formation of a phosphodiester bonds
• DNA ligases require ATP for function
• The E. coli ligase joins only double stranded DNA, while the
bacteriophage T4 ligase can also join double stranded RNA
or RNA/DNA hybrids

You order two reverse complementary oligos – after

annealing (how?), you want to ligate them together to
generate concatemers by adding T4 ligase and ATP,
but doesn’t work – why?
 Polymerases
• RNA-dependent RNA polymerase (not frequently used in
molecular biology applications)
• DNA-dependent RNA polymerase (RNAP, 3 in vertebrates,
five in plants – used for making RNA in in vitro transcription
experiments, often derived from viruses; e.g., T7, SP6)
• DNA-dependent DNA polymerase – Often used to copy or
fill-in DNA – includes E. coli DNA Pol I (has also 5’3’ and
3’5’ exonuclease activities – why?), we often use the large
Klenow fragment that lacks the 5’3’ exonuclease activity
(why?), thermostable DNA polymerases (e.g., Taq)
• RNA-dependent DNA polymerase – a.k.a. reverse
transcriptase
 Classical (First Generation) Sequencing

Sanger Maxam & Gilbert

Heather & Chain, 2016

Genomics 107:1-8
 Cloning
• Molecular cloning is a set of techniques used to insert and
modify recombinant DNA from a prokaryotic or eukaryotic
source into a replicating vehicle, a vector
• Vectors are usually propagated in a host, and Escherichia coli
(E. coli) is one of the most commonly used bacterial hosts

Vector Types
Plasmids (< 6 kbp) – Examples: pBR322, pUC
Phagemids (< 3 kbp) – Example: pBSK
Phages (< 22 kbp) – Example: λ, M13, T7, T3
Cosmids (< 45 kbp)
Yeast Artificial Chromosomes (YACs, < 100 kbp)
Bacterial Artificial Chromosomes (BACs, < 350 kbp)
 Choosing a vector
1) What is the size of the inserts you want to clone?
A cDNA library will have inserts around 1 – 2 kbp; for a
genomic DNA library, the longer the better (> 30 kbp)

2) What is the purpose of a particular cloning?

Protein expression, generation of large quantities of DNA
(keep in mind copy number), sequencing a genome

3) Appropriate combination of host and selectable

marker
What is a library?
 Plasmids

~4.8 x106 bps

3 – 10 x103 bps
 Plasmids
• Extrachromosomal, double stranded
circular DNA present in most bacteria
• Relaxed replication (as opposed to
stringent), meaning that it is
independent of the replication of the
bacterial genome
• Medium copy number (~20 per cell) –
can be increased with chloramphenicol
30-50 times
• Many modern plasmids (e.g., pUC)
derived from pBR322. Most carry
mutation in ori that increase number to
500 – 1,000 copies/cell
Note:
Origin of replication (ori)
Selectable markers (amp, tet)
Restriction sites for cloning
Antibiotics and how they work
 Resistance to antibiotics

Penicillin core

β-lactamase (encoded by AmpR markers)

• Penicillin derivatives include Ampicillin and Amoxicillin

• Resistance to other antibiotics involve either modification of
the site of action, or degradation (like for penicillin)
• Frequently resistance provided by plasmids that can be
exchanged between bacteria
 Modern Plasmids

• Small size allows larger inserts

(<10 kb)
• High copy number
• Presence of a multi-cloning site
(many unique restriction enzyme
sites)

through 𝛂-complementation
• Provides blue/white selection

Essential components of plasmids are the origin of replication (ori) and a

selectable marker (e.g., resistance to an antibiotic)
 Bacteriophages (or just phages)
• Phages are viruses of bacteria, and there are many
different ones
• ”Bacteriophage" means "bacteria eater"
• Phages encode some enzymes necessary for
replication and packaging, but mainly use the
cellular machinery for metabolites and enzymes
• Depending on the environment and host growth
conditions, they can choose to lyse the host (lytic
cycle), or integrate into the genome and wait for a
better time (lysogenic cycle) – some can only do
lysis
 λ Phage

• λ DNA is double stranded (~48.5 kbp) carried as a linear molecule

with 12 nucleotide single-stranded DNA cohesive termini (the cos
sites)
• A fascinating molecular switch mechanism permits λ to switch from
lytic to lysogenic growth (prophage), and vice versa (we will talk
more about this next week)
• Used as a vector by insertion of DNA (< 5 kbp) or by replacement
(< 22 kbp) – many variants available depending on need
Credits: digital.wwnorton.com; https://bioinfo2010.wordpress.com/
 λ Phage

• λ uses the maltose receptor to get into E. coli cells – in the lab, you
grow E. coli in media containing maltose to make them more
receptive to λ infection
• Once inside the cell, the double stranded λ linear DNA circularizes
through annealing of the complementary cos sites (cohesive ends)
λ Phage- Lytic Cycle
 λ Phage- Lytic Cycle
Lysogenic
Phage plaque

Bacterial lawn Lytic

λ as a cloning vector

bio3400.nicerweb.com
Bacteriophage Plaque Formation Assay

Obtained from https://digital.wwnorton.com/ebooks/epub/microbio4/OEBPS/images/sfmb4e_0638.jpg/

Cloning Without the Pain of Ligations

Karimi et al, 2007, Plant Physiol 145: 1144-1154

 Molecular Cloning Steps

But vector B can potentially re-

ligate without fragment A

https://www.neb.com/

How do you discriminate between re-ligated

vector and plasmid containing the insert?
 Analysis of Nucleic Acids: Visualization
• Nucleic acids (DNA or RNA) can be easily separated by
electrophoresis
• Agarose electrophoresis for larger fragments (80 – 100 bp or
more) – can be preparative
• Polyacrylamide electrophoresis for smaller (~10 bp to ~800 bp)
• Native
• Denaturing (urea)
• Nucleic acids can be detected by:
• Ethidium bromide
• Limit ~5 – 10 ng of DNA, much more efficient on dsDNA
• Blotting to solid support and hybridization to labeled probe (e.g.,
fluorescent, radioactive)
• DNA – called Southern blotting
• RNA – called Northern blotting
RNA has rich secondary structure – how do
you separate by size for a Northern?
 Analysis of Nucleic Acids: Quantitation
• Nucleic acids can be easily quantitated by spectrophotometry –
resonance of aromatic cycles of purines and pyrimidines provides
good absorbance at 260 nm – estimated at 1 OD for 50 ng/μl for
DNA (~ 1 OD for 40 ng/μl for DNA)
• Purity of DNA or RNA can be easily estimated by the A 260/A280 ratio
– a value of 1.8 is considered good for DNA and 2.0 for RNA
What absorbs at 280 nm?
• A number of methods of nucleic acid quantification are based on
using fluorophores that fluoresce when bound to DNA (e.g., Qubit)
– much more sensible than UV absorbance (Qubit limit ~0.2 ng/μl –
this would give you A260 = 0.04 – tough to believe)
• I personally favor quantification by agarose electrophoresis
comparing to known standards when possible
 Analysis of Nucleic Acids: Sequencing
• First generation DNA sequencing methods include Maxam & Gilbert
and dideoxy (Sanger) chain termination methods
• Next generation sequencing methods include a number of different
techniques:
 Classical (First Generation) Sequencing

Sanger Maxam & Gilbert

Heather & Chain, 2016

Genomics 107:1-8
The Cost of Sequencing Continues to Drop
Questions?