Lezione 23 - 24 martedì 10 maggio 2011

1. Lezione 23 - 24martedì 10 maggio 2011 corso vettori biologici II Biotec industriali ore 14:00 -16:00 aula 6A

2. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting (Nucleic Acids Research, 2011, 1ﾐ11 doi:10.1093/nar/gkr218) TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.

3. Nuovo metodo di assemblaggio A Modular Cloning System for Standardized Assembly of Multigene Constructs (PLoS One. 2011 Feb 18;6(2):e16765) The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.

4. The Golden Gate assembly method (and MoClo) The Golden Gate method (Engler 2008, Engler 2009, Engler 2011) offers standardized, quasi-scarless, multi-part DNA assembly, and is an excellent choice for combinatorial library construction (see also a variation of the Golden-gate protocol for use with j5). The Golden Gate method relies upon the use of type IIs endonucleases, whose recognition sites are distal from their cut sites. Although there are several different type IIs endonucleases to choose from, the example below uses BsaI (equivalent to Eco31I) (the Golden Gate method only uses a single type IIs endonuclease at time).

5. Golden Gate assembly of partA with a linearized destination vector:

6. Golden Gate assembly put together

7. Golden gatecombinatory library

8. Cloning versus syntetic Generation of families of construct variants using golden gate shuffling. Methods Mol Biol. 2011;729:167-81. Current standard cloning methods based on the use of restriction enzymes and ligase are very versatile, but are not well suited for high-throughput cloning projects or for assembly of many DNA fragments from several parental plasmids in a single step. We have previously reported the development of an efficient cloning method based on the use of type IIs restriction enzymes and restriction-ligation. Such method allows seamless assembly of multiple fragments from several parental plasmids with high efficiency, and also allows performing DNA shuffling if fragments prepared from several homologous genes are assembled together in a single restriction-ligation. Such protocol, called Golden Gate shuffling, requires performing the following steps: (1) sequences from several homologous genes are aligned, and recombination sites defined on conserved sequences; (2) modules defined by the position of these recombination sites are amplified by PCR with primers designed to equip them with flanking BsaI sites; (3) the amplified fragments are cloned as intermediate constructs and sequenced; and (4) finally, the intermediate modules are assembled together in a compatible recipient vector in a one-pot restriction-ligation. Depending on the needs of the user, and because of the high cloning efficiency, the resulting constructs can either be screened and analyzed individually, or, if required in larger numbers, directly used in functional screens to detect improved protein variants. Engler C, Marillonnet S.

9. Un nuovo codice genetico !!! The most distinctive characteristic of TAL effectors is a central repeat domain containing between 1.5 and 33.5 repeats that are usually 34 residues in length (the C-terminal repeat is generally shorter and referred to as a “half repeat”).[2] A typical repeat sequence is LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG, but the residues at the 12th and 13th positions are hypervariable (these two amino acids are also known as the repeat variable diresidue or RVD). The experimentally validated code between RVD sequence and target DNA base can be expressed as (asparagina-isoleucne) NI = A, (istidina-aspart ac)HD = C, (asparagina-glycine)NG = T, (aspar-asparag)NN = G or A, and (aspar-serine)NS = A, C, G, or T. Further studies has shown that the RVD (asparag-lysine)NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5’ base targeted by the first repeat, but the exact mechanism of this recognition is not known.

10. Sintetizzare nuovi effettori TAL This simple code between amino acids in TAL effectors and DNA bases in their target sites might be useful for protein engineering applications. Numerous groups have design artificial TAL effectors capable of recognizing new DNA sequences in a variety of experimental systems. Such engineered TAL effectors have been used to create artificial transcription factors that can be used to target and activate endogenous genes in tomato, Arabidopsis thaliana, and human cells. Engineered TAL effectors can also be fused to the cleavage domain of FokI to create TAL effector nucleases (TALENs) that show activity in a yeast-based assay, at endogenous yeast genes, in a plant reporter assay, at an endogenous plant gene, and at endogenous human genes. The human HPRT1 gene has been targeted at detectable, but unquantifiable levels and TALENs containing the FokI cleavage domain fused to a different portion of the TAL DNA binding domain have been used to target the endogenous NTF3 and CCR5 genes in human cells with efficiencies of up to 25%. Engineered TAL effectors can be constructed using either conventional gene synthesis or modular assembly. TAL effector nuclease constructs are also commercially available from a European biotechnology company. Such nucleases share some properties with zinc finger nucleases and may be useful for genetic engineering and gene therapy applications

11. Talen rec system One of the pairs of TALENs targeting the human HPRT1 gene was subcloned into the mammalian expression vector pCDNA3.1(-) (Invitrogen) using XhoI and AflII. These enzymes excise the entire TALEN from pTAL3 or pTAL4 and place the coding sequence under control of the CMV (cytomegalovirus) promoter. The resulting plasmids were introduced into HEK293T cells by transfection using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Cells were collected 72 h after transfection and genomic DNA isolated and digested with Hpy188I, which cuts in the spacer sequence of the TALEN target site. After digestion, a chromosomal fragment encompassing the target site was amplified by PCR. Upon completion, the reactions were incubated for 20 min at 72 C with 4 ml of Taq DNA poly-merase. PCR products then were digested with Hpy188I and cloned in a TOPO TA vector (Invitrogen) Independent clones containing the full-length PCR product were sequenced to evaluate mutations at the cleavage site.