Type IIS Restriction Enzymes Quiz

Genome Editing: Generating Mutants using CRISPR-Cas9

• Cloning sgRNAs into plant expression vector is a key part of creating mutants using CRISPR-Cas9.
• Golden Gate cloning is a technique used for this process.
• Type IIS restriction enzymes are also used in the process.
• E. coli transformation is part of the procedure.

Common Gene Cloning Techniques

• Traditional cloning using type IIP restriction enzymes (digestion and ligation) is a common technique.
• Golden Gate assembly (or type IIS assembly) is another common method.
• TA and blunt end cloning are also included in the set of common techniques
• Recombinational cloning (Gateway) technology is a cloning technique
• Gibson assembly is part of the set of common techniques

Cloning sgRNAs in Plant Expression Vector

• ChopChop is used in the cloning process, which relates to sgRNAs.
• CBF4-sg11 and CBF4-sg1 are used together, with sgRNAs.
• PCBC-DT1T2 CRISPR scaffolds are used with Hot-Start Phusion Pol.
• PCR is used in the process to generate the desired PCR product
• pHEE401E vectors used with Bsal, and T4 DNA Ligase.
• sg11-1 cbf4/ pHEE401E is the final CRISPR expression Vector produced with this process.

Designing Primers with cbf4-gRNAs

• Primers CBF4-sg11_For and CBF4-sg1_Rev are essential for the process.
• The prime sequence for CBF4-sg11_For has overhangs with Bsal sites.
• The primer sequence for CBF4-sg1_Rev also has overhangs with Bsal sites, so the Bsal sites are also present in CRISPR vector.

Overview of cloning strategy

• PCR product amplified with sg11+sg1 is part of the cloning strategy.
• CRISPR vector (pHEE401E) used for cloning gRNA cassette.
• KanR/SpecR, BastaR expression cassettes with CRISPR vector, used for the cloning strategy.
• Final cbf4-CRISPR vector (cbf4 sg11+1/pHEE401E) used next
• The cloning strategy utilizes Bsal, SpecR, U6, and sgRNA.

PCR Amplification using Hot-Start Phusion DNA Polymerase

• Phusion High-Fidelity DNA Polymerase is essential for amplification.
• This DNA polymerase has high processivity and extremely low error rates, making it appropriate for cloning.
• Hot Start Phusion polymerase inhibits activity at room temperature to prevent nonspecific products.
• The polymerase has 5' to 3' polymerase and 3' to 5' exonuclease activities, resulting in blunt-ended products
• The error rate is significantly lower than that of Taq and Pyrococcus furiosus DNA polymerases.

Golden Gate cloning with Type IIS Restriction Enzymes

• Type IIS restriction enzymes (REs), such as Bsal and others, cut DNA a defined distance from their recognition sequence.
• Golden Gate cloning using Type IIS REs is crucial for sequence independent cloning of genes.
• This technique is a single-tube, one-step process involving digestion and ligation in the same tube.
• The technique eliminates scar sequences, and allows simultaneous assembly of multiple fragments.

Type II Restriction Enzymes

• Restriction enzymes are classified according to whether the cleavage occurs within or outside the recognition site (Type II or II respectively).

Type IIP Restriction Enzymes

• Classical Type IIP REs (e.g., EcoRI, BamHI) are highly characterized.
• These enzymes recognize specific 4-8 nucleotide palindromic sequences.
• They cleave within the recognition site, yielding sticky or blunt ends.

Type IIS Restriction Enzymes

• Type IIS enzymes (Bsal, Bbsl, etc.) cleave DNA at a distance from the recognition sequence.
• The catalytic and recognition domains are separated by polypeptide linkers.
• The sequences beyond the recognition site can contain any combination of nucleotides.
• This cloning technique allows assembly of multiple fragments with complementary overhangs using Golden Gate assembly.

How does Golden Gate cloning work?

• Type IIS restriction enzymes cut DNA downstream of their recognition sites.
• Fragments with unique overhangs are assembled by annealing complementary four-base overhangs
• Restriction sites are removed from the final ligation product, enabling simultaneous digestion and ligation.

Cloning a Single Fragment using Type IIS Restriction Enzymes

• The DNA fragment can be a PCR product or cloned PCR product.
• Type IIS recognition sites on the fragment are oriented so cleavage leaves sticky ends but removes enzyme binding sites
• Recipient vector must have matching Type IIS sites to allow for ligation.

Cloning Multiple Fragments using Type IIS Restriction Enzymes

• Each fragment has unique overhangs, dictating the assembly order.
• Complementary overhangs facilitate the assembly onto adjacent fragments in the final assembly.
• T4 ligase joins complementary overhangs.

Ligation with T4 DNA Ligase

• T4 DNA ligase forms phosphodiester bonds between 5' phosphate and 3' hydroxyl groups.
• This process is equivalent to repairing nicks in a duplex DNA molecule.
• In recombinant DNA technology, DNA ligase joins restriction fragments using ATP.
• Bacteriophage T4 DNA ligase is the preferred enzyme for joining both sticky and blunt ends.

Golden Gate Reaction

• The digestion and ligation are performed in the same reaction using Bsal and T4 DNA ligase.
• Specific buffers, ATP, and Mg²⁺ are included in the reaction.
• The reaction is performed in a thermal cycler with specific temperature cycles for digestion and ligation.

Golden Gate Cloning: Summary

• Golden Gate utilizes Type IIS enzymes for one-step DNA fragment insertion into a vector.
• The technique repeatedly cycles between restriction endonuclease and DNA ligase temperatures
• Golden Gate reduces the number of steps in conventional cloning procedures.

E. coli Transformation

• E. coli transformation is a natural process where bacteria take up foreign DNA.
• Molecular biology techniques make bacteria competent for taking up DNA using methods like Calcium chloride.
• Transformation occurs when competent cells are mixed with the DNA, heat shocked, and allowed to recover.
• Antibiotic selection identifies successfully transformed cells.

Plasmids

• Plasmids are circular, double-stranded DNA that replicate independently of the bacterial chromosome.
• Essential elements of plasmids include an origin of replication and an antibiotic resistance gene.

Plasmid Elements: ori

• ori is a short DNA sequence crucial for directing plasmid replication.
• This region recruits the DNA replication machinery inside the bacteria, making copying (replication), possible.

Expression Vector

• Expression vectors are used to study gene function by introducing a gene’s DNA fragment.
• An expression vector has three components: a promoter, a DNA fragment (eg, CDS/ORF), and a terminator.

Delivery of sgRNA in Plants by Agrobacterium tumefaciens

• Agrobacterium tumefaciens is used to deliver sgRNA to plants.
• This method involves cloning into a plant expression vector, transforming E. coli, and introducing the transformed plasmid into Agrobacterium culture.
• Then the Agrobacterium culture then transforms the target plant, selecting transformed plants for further study through germination and harvest of seeds.

Test your knowledge on Type IIS restriction enzymes and their applications in cloning techniques. This quiz covers features, recognition sites, and the significance of unique overhangs generated during the Golden Gate cloning process. Challenge yourself with questions about their function and usage in genetic engineering.