3A assembly (three antibiotic assembly) is a method for assembling two BioBrick parts (standardized DNA parts) and selecting for correct assemblies with antibiotics. 3A assembly is now the preferred assembly method at iGEM HQ and the Knight lab at MIT.
3A Assembly relies on three way ligation (between the two parts and the backbone vector) for assembly. It uses effective antibiotic selection to eliminate unwanted background colonies and eliminates the need for gel purification and colony PCR of the resulting colonies. Note that this method does produce scars as prefixes and suffixes (restriction sites flanking the BioBrick) is used. This produce 6 or 8 bp of “scar” (empty sequence) between the 2 BioBrick ligated.
The following are the procedures and corresponding importance of the steps
Gibson Assembly is a cutting-edge DNA ligation technique developed by Dan Gibson at JCVI in 2009.
It uses three enzymes to ligate two or more sequences of DNA when they have overlapping end sequences at their joining point (~40bp). These overlapping regions can be easily added to the ends of any length of DNA by using PCR with primers which have added "flaps". Thus PCR followed by Gibson allows you to join any two blunt ended pieces of DNA.
Gibson Assembly master mix contains 3 enzymes:
The Gibson reaction relies on the action of the T5 exonuclease - this chews back at the 5' ends of both pieces of DNA.
Once it has chewed back far enough A-T G-C base pairing allows the two pieces to bind together.
We now have a single piece of DNA but it is not physically ligated together, it is merely held together by hydrogen bonding, also there are gaps in both single strands.
Phusion is a DNA polymerase that repairs these gaps. It extends from the 3' end, so it does not interfere with T5 exonuclease which is acting at 5' ends.
Now we have DNA with no missing fragments but there is still a break in the phosphodiester bonds in the backbones of both single strands of DNA. This is corrected when Taq ligase action forms this bond.
And finally we have our finished piece of DNA
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a non-specific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone non-homologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of CRISPR/Cas-based RNA-guided DNA endonucleases.
ZFNs are fusions of the non-specific DNA cleavage domain from the FokI restriction endonuclease with zinc-finger proteins. ZFN dimers induce targeted DNA double-strand breaks (DSBs) that stimulate DNA damage response pathways. The binding specificity of the designed zinc-finger domain directs the ZFN to a specific genomic site.
TALENs are fusions of the FokI cleavage domain and DNA-binding domains derived from TALE proteins. TALEs contain multiple 33–35 amino acid repeat domains that each recognizes a single base pair. Like ZFNs, TALENs induce targeted DSBs that activate DNA damage response pathways and enable custom alterations.
CRISPR are loci that contain multiple short direct repeats, and provide acquired immunity to bacteria and archaea. CRISPR systems rely on crRNA and tracrRNA for sequence-specific silencing of invading foreign DNA. Three types of CRISPR/Cas systems exist: In type II systems, Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA-tracrRNA target recognition.
Mechanism of ZFN, TALEN and CRISPR/Cas9. A. ZFN, TALEN and CRISPR/Cas9 achieve precise and efficient genome modification by inducing targeted DNA DSBs, which would be corrected by NHEJ and HR repair mechanisms. NHEJ-mediated repair leads to the introduction of variable length insertion or deletion. HR-mediated repair could lead to point mutation and gene replacement, in the present of donor DNA. B. TALEs and Cas9 protein fused with effector proteins such as VP64, Mxi1 could regulate expressions of endogenous genes.
Additionally, TALEs fused with histone-deacetylating epigenetic effectors could regulate epigenetics status of endogenous genes. CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; dCas9, inactive Cas9 protein; DSB, Double Strand Breaks; NHEJ, Error-prone Nonhomologous End Joining; HR, Homologous Recombination; InDel, Insertion and Deletion; PAM, Protospacer Adjacent Motif; RNA Pol II, RNA Polymerase II; sgRNA, single guide RNA; TALE, Transcription Activator-Like Effector; TALEN, Transcription Activator-Like Effector Nuclease; ZFN, Zinc Finger Nuclease.
Advances in genome editing technology and its promising application in evolutionary and ecological studies - Scientific Figure on ResearchGate.