Workshop 1 USER Teis Solution

Applied Molecular Cellular Biology

🧬 Applied Workshop 1 — Complete Theoretical Summary (Exam-Ready)

This workshop combines molecular cloning theory, PCR design, USER cloning, DNA repair mechanisms, and exam strategy. The goal is not just to know what to do, but why each step is done.

1️⃣ Agrobacterium-mediated transformation 🌱

What is Agrobacterium?

Agrobacterium is a soil bacterium naturally attracted to plant cells.
It recognizes plant-derived signals (hormones, sugars) indicating nearby plant tissue.
Once attached, it activates genes on its Ti plasmid.

Natural infection mechanism

Agrobacterium senses plant signals
Activates plasmid genes
Produces transport proteins
Transfers DNA into plant cells
Induces uncontrolled plant cell division (tumor/crown gall formation)

Why is this useful?

The DNA transfer system is hijacked for biotechnology
Scientists can replace the bacterial genes with genes of interest
Used for:
- 🌱 Plant transformation
- 🍄 Fungal transformation

➡️ Key idea: Agrobacterium is a natural genetic engineer that we reprogram.

2️⃣ DNA double-strand breaks & repair mechanisms 🧬

Why DNA repair matters

Double-strand breaks (DSBs) are lethal if unrepaired
Cells have two major repair pathways

🔹 Non-Homologous End Joining (NHEJ)

Fast but error-prone
Sticky ends are trimmed and ligated
Often loses nucleotides
Can cause:
- Frameshifts
- Non-functional proteins

📌 Important exam note:

Only ~1% of the genome encodes proteins → many breaks occur in non-coding DNA

🔹 Homologous Recombination (HR)

High-fidelity repair
Uses a homologous DNA template
Common in:
- Diploid organisms (e.g. humans)
- Yeast and fungi

Why HR is critical for cloning

Researchers exploit HR to:
- Insert DNA precisely
- Replace genes
- Create knockouts or overexpression strains

➡️ This is the biological basis of USER cloning

3️⃣ USER cloning — core concept 🧪

What does USER stand for?

Uracil-Specific Excision Reaction

Commercial system (e.g. NEB)
Exact enzyme mix is proprietary
Generates defined overhangs for seamless cloning

Why use USER cloning?

No ligase required
Highly directional
Multiple fragments assembled simultaneously

⚠️ Nickname in lab: “USER-unfriendly cloning” → powerful but technically demanding

4️⃣ Fragment design in USER cloning 🧩

A typical construct uses four fragments:

Fragment	Function
1	Left homologous border
2	Right homologous border
3	Selection marker (e.g. antibiotic resistance)
4	Vector backbone

📌 Fragments 1 & 2 enable homologous recombination 📌 Fragment 3 allows selection 📌 Fragment 4 maintains plasmid integrity

5️⃣ Primer design for homologous borders ✏️

Key principles

Borders must be long:
- Typically 300–1000 bp (fungi/USER system)
Primers must:
- Amplify genomic DNA
- Include USER overhangs
- Be oriented correctly (forward + reverse)

Why genomic DNA (not cDNA)?

Borders must lie outside the gene
cDNA lacks introns and flanking regions
Therefore genomic DNA is mandatory

6️⃣ DNA polymerases — choosing the right one ⚙️

Polymerases discussed

Polymerase	Proofreading	Use case
Cheap Taq-like	❌	Fast screening
Fusion Hot-Start	✅	High-fidelity cloning
Pfu Turbo Cx	✅ + Uracil-tolerant	USER cloning

Why Pfu Turbo Cx?

USER cloning requires uracil in primers
Most polymerases stall at uracil
Pfu Turbo Cx is engineered to bypass uracil

➡️ Exam gold: Always use proofreading polymerases for cloning

7️⃣ Hot-start PCR — why it matters 🔥

What is hot-start?

Polymerase inactive at room temperature
Activated only after heating (~95°C)

Advantages

Prevents:
- Primer-dimers
- Non-specific amplification
Crucial when:
- Setting up many reactions
- Working slowly at bench

8️⃣ PCR program — theoretical logic 🧠

Typical PCR steps

Initial denaturation
- ~94–98°C
Denaturation
- ~90–98°C
Annealing
- Depends on primer Tm (≈50–68°C)
Extension
- 72°C
- ~1 min / kb
Cycles
- Usually 20–34

Why not 50–80 cycles?

Enzymes degrade
dNTPs become limiting
Non-specific products accumulate

📌 Rule of thumb:

Each extra cycle ≈ doubles DNA
4 extra cycles = 16× more DNA

9️⃣ Agarose gel electrophoresis 🧫

Gel components

Agarose (typically 1%)
TAE buffer
Loading dye
Ethidium bromide

Loading dye functions

Adds density (sample sinks)
Adds tracking colors
Allows visual monitoring of run

Ethidium bromide ⚠️

Intercalates DNA → fluoresces under UV
Mutagenic
Handle with gloves
Waste disposed in sealed plastic, not accumulated

🔟 Restriction enzymes in USER cloning 🔪

Enzymes mentioned

EcoRI
BamHI
HindIII
PacI (important)
Nb.BbvCI

Key idea

USER vectors are opened with PacI
Nb.BbvCI generates USER-compatible overhangs
These match the overhangs added via PCR primers

📌 Enzymes that don’t contribute are excluded

1️⃣1️⃣ USER enzyme activity

USER mix contains endonucleases
Cuts within DNA
Exonucleases would cut from ends (not desired)

1️⃣2️⃣ USER cloning vs CRISPR-Cas ⚔️

USER cloning

Technically complex
Multi-fragment assembly
Educational for molecular biology principles

CRISPR-Cas

Faster
More precise
PCR primers include homology arms
DNA inserted directly with Cas9

➡️ Modern labs increasingly prefer CRISPR

1️⃣3️⃣ Exam structure & strategy 📚

Exam format

Digital exam
No internet
No personal notes
Word + drawing tools allowed

Paper-based exam preparation

Paper released 48 hours before exam
Exam tasks:
- 40–50% paper understanding
- 50–60% experimental design

Typical questions

Summarize paper idea
Apply methods to new scenario
Design experiments
Interpret figures

➡️ Reasoning matters more than “correct” answers

1️⃣4️⃣ Experimental reasoning (food contamination examples) 🍔

Fungal contamination

Possible solutions:

Lower temperature
Anaerobic conditions
Adjust light
Add beneficial microbes
Genetic engineering (GMO)

Engineered bacteria design (theory)

Plasmid must contain:

Bacterial origin of replication
Antibiotic resistance marker
Promoters (temperature-regulated)
Toxic gene against fungi
Terminators

1️⃣5️⃣ Detection methods (theoretical toolbox) 🔍

PCR
ELISA
Sequencing
Proteomics
Southern / Northern / Western blot
Immunoassays

PCR essentials

Template DNA
Primers
Polymerase
dNTPs
Buffer (Mg²⁺ critical)

🎯 Final exam take-home messages

Explain your reasoning
There may be multiple valid answers
Demonstrate understanding, not memorization
Connect molecular tools to biological logic

Quiz

Score: 0/29 (0%)

Q0. What is the primary reason Agrobacterium is useful for plant and fungal genetic engineering?

It naturally kills plant cells

It can naturally transfer DNA into host cells

It replicates faster than other bacteria

It only infects fungi

Q1. Which DNA repair mechanism is exploited during USER cloning to integrate DNA into the genome?

Non-homologous end joining

Base excision repair

Homologous recombination

Mismatch repair

Q2. Why is non-homologous end joining considered error-prone?

It requires a homologous template

It introduces random mutations intentionally

It often removes nucleotides at break sites

It only occurs in coding regions

Q3. What does USER stand for in USER cloning?

Universal Sequence Excision Reaction

Uracil-Specific Excision Reaction

Unified Strand Exchange Reaction

Universal Site Enzyme Recombination

Q4. Which fragments in USER cloning provide homology for recombination?

Fragment 3 and 4

Fragment 1 and 2

Fragment 2 and 3

Fragment 1 and 4

Q5. Why must genomic DNA be used instead of cDNA when designing homologous borders?

cDNA is unstable

Genomic DNA contains introns and flanking regions

cDNA cannot be amplified by PCR

Genomic DNA has higher GC content

Q6. Which DNA polymerase is specifically suitable for USER cloning?

Standard Taq polymerase

Fusion Hot-Start polymerase

Pfu Turbo Cx polymerase

RNA polymerase

Q7. What is the main purpose of a hot-start DNA polymerase?

Increase extension speed

Prevent enzyme degradation

Reduce non-specific amplification

Allow RNA amplification

Q8. Why are PCR reactions typically limited to around 20–34 cycles?

Primers degrade after 34 cycles

DNA polymerase becomes inaccurate after many cycles

Enzymes and dNTPs become limiting

PCR machines cannot run longer programs

Q9. What is the function of loading dye in agarose gel electrophoresis?

To stain DNA permanently

To cut DNA into fragments

To add density and track migration

To protect DNA from UV light

Q10. Which property makes ethidium bromide useful in gel electrophoresis?

It digests DNA

It fluoresces when bound to DNA

It stabilizes DNA structure

It increases DNA migration speed

Q11. Which restriction enzyme is critical for opening USER vectors?

EcoRI

BamHI

HindIII

PacI

Q12. What type of enzyme activity is present in the USER enzyme mix?

Exonuclease activity

Ligase activity

Endonuclease activity

Reverse transcriptase activity

Q13. Why has CRISPR-Cas largely replaced USER cloning in many laboratories?

USER cloning no longer works

CRISPR is cheaper and simpler

CRISPR requires no PCR

USER cloning cannot be used in fungi

Q14. What is the main learning objective of including USER cloning in teaching despite newer technologies?

It is industry standard

It teaches fundamental molecular biology principles

It is legally required

It is cheaper than CRISPR

Q15. Homologous recombination requires a homologous DNA template.

True

False

Q16. Non-homologous end joining always leads to gene mutations.

True

False

Q17. USER cloning can assemble multiple DNA fragments in a single reaction.

True

False

Q18. cDNA is preferred over genomic DNA for designing homologous borders.

True

False

Q19. Proofreading activity in DNA polymerases increases cloning accuracy.

True

False

Q20. Hot-start polymerases are active at room temperature.

True

False

Q21. One minute of PCR extension typically synthesizes about 1 kb of DNA.

True

False

Q22. Ethidium bromide should be handled carefully because it is mutagenic.

True

False

Q23. USER cloning relies on ligase to join DNA fragments.

True

False

Q24. PacI is used because it generates compatible ends for USER overhangs.

True

False

Q25. CRISPR-Cas requires homologous repair templates for precise insertions.

True

False

Q26. Exam questions always have a single correct experimental solution.

True

False

Q27. Experimental design questions test understanding rather than memorization.

True

False

Q28. Beneficial bacteria can be used to outcompete fungi in food fermentation.

True

False