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While every effort has been made to follow citation style rules, there may be some discrepancies. DpnII and TaqI are 4 base cutters, specifically targetting and cutting the sequences GATC and TCGA respectively. A distinct advantage of using restriction enzymes for our 'killing' mechanism is that the the genetic material is removed, but the cell membrane is left intact. This involves the size analysis of restriction fragments produced by several restriction enzymes individually and in combination. The precise usage of codons, information regarding mutations and polymorphisms and identifica­tion of gene regulatory control sequences can only be elucidated by analyzing DNA sequences. In this the reaction mixture is divided into four groups, representing the four dNTPs A, C, G and T. Since the incorporation of ddNTP rather than dNTP is a random event, the reaction will produce new molecule varying widely in length, but all terminating at the same type of base.
Since every band in the track from dideoxyadenosine triphosphate must contain molecules that terminate at adenine, and those in ddCTP terminate at cytosine, etc., it is possible to read the sequence of the newly synthesized strand from autoradiograph, provided that the gel can resolve differences in length equal to single nucleotide. It is possible to undertake nucleotide sequencing from double-stranded molecules such as plasmid cloning vectors and PCR products but the double-stranded DNA must be denatured prior to annealing with primer. Denaturants such as form amide or dimethylsulphooxide (DMSO) are usually em­ployed to prevent renaturation of PCR strands after their separation it is possible to physically separate and retain one PCR strands by incorporating a molecule such as biotin into one of the primer, which can be recover after PCR by affinity chromatography with streptavidin, leaving the complimentary PCR strand.
This involves dideoxynucleotides labelled with different flurochromes and are used to carry out chain termination as in standard re­actions. Each product with their base specific dye is excited by a laser and dye then emits light at its characteristic wavelength. This is a chemical method of sequencing developed by Maxam and Gilbert and the method is often used for sequencing of small fragments of DNA such as oligonucleotides. DNA labelled at one end is divided into four aliquots; each is treated with chemicals that act on specific bases by methylation or removal of bases.
After modification reactions separate samples are cleaved by piperidine, which breaks phosphodiester bonds exclusively at the 5′-side of nucleotides whose base has been modified. Our Sexy Plant is a challenging project for many reasons; a very important one is that we use plants as chassis for engineering. As BioBricks, GB is a modular cloning strategy that allows the fabrication of new devices by the combination of prefabricated standard modules.
Type IIS restriction enzymes, unlike type II enzymes; cleave DNA at a defined distance from their recognition sites, not requiring any specific sequence in the cleavage site. The first step in the GB cloning strategy is the adaptation of the DNA sequence to the GB standard. There are three basic categories that define the most common parts making up a transcriptional unit.
The mutagenesis procedure required to remove internal restriction sites is standardized and involves the amplification of the target DNA in separated fragments (GBpatches) using GB-adapted primers, which incorporate single mismatches to disrupt the enzyme target sites.
Domesticated GBparts can now be assembled together in a one-tube-one-step reaction to create a Transcriptional Unit (TU). Multigene constructs are important in Plant Synthetic Biology because they allow several TUs to be jointly transferred to the plant genome. GB-made TUs can be combined using GB binary assemblies to create increasingly complex multigenic structures.


The double loop topology of the GB cloning strategy allows an indefinite growth of the constructs through iterative binary assembly steps.
Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes.
One type of cut produces two DNA strands with blunt ends; that is, there is no overhang on one strand or the other. This leads to multiple double-stranded breakages in DNA, which are unlikely to be repaired in time and will subsequently result in cell death. Two techniques have been developed for this, one based on an enzymic method frequently termed Sanger sequencing and chemical method called Maxam and Gilbert sequencing. Thus four sets of DNA sequences are generated, each terminating at a different type of base, but all have a common 5′- end. Very thin and long gels are used for maximum resolution over a wide range of fragment lengths. This is not strictly a PCR, since it involves linear amplification with a single primer in about 20 PCR cycles.
The advantage of this modification is that, since different labellel is incorporated in each ddNTP all the products are run on same denaturing electrophoresis gel. A diffraction grating separates the emissions, which are detected by charge couple device (CCD) and the sequence is interpreted by computer. Conditions are chosen such that each molecule is modified at only one position along its length and every base in the DNA strand has equal chances of being modi­fied.
The result is similar to that produced by Sanger method, since each sample now contains radiolabelled molecules of various length, all with one end common (the labelled end), and with the other end cut at the same type of base.
Plants have eukaryotic gene structure, make use of plant-specific regulatory regions and require special T-vectors for transformation, among other special features. A difference between both strategies is that BioBricks is based on type II enzymes and GB relies on the use of type IIS restriction enzymes. Since there are no sequence requirements in the cleavage sites, these can be defined by the user and adapted to serve as standard fusion sites to DNA parts. These part categories are PROM (GGAG-AATG), CDS (AATG-GCTT) and TER (GCTT-CGCT) and were the most used on this project. Each primer pair is used to amplify a GBpatch by PCR, and the resulting fragments are assembled together in a BsmBI restriction-ligation reaction into pUPD.
The special orientation and disposition of the restriction sites in the GB destination vectors defines two levels of destination plasmids: the ? plasmids used for BsaI reactions and the ? plasmids, which are used for BsmBI GB reactions. By choosing the appropriate combinations of expression and destination vectors, increasingly complex multigenic modules are created. A second type of cut produces two strands with sticky ends; because of the site of cleavage, each strand extends beyond the complementary region of the strand pair. If the resultant fragments are 2 kb, 3 kb and 4 kb away, then A and B cut at opposite ends of the molecule; if they are 1 kb, 2 kb and 6 kb away the sites are near to each other. The four labelled and chain-terminated samples are then denatured by heating and loaded next to each other for electrophoresis. After electrophoresis, the position of radioactive DNA bands on the gel is determined by autoradiography.
Radiolabeled or fluores­cent—labelled dideoxynucleotides are then introduced into the final stages of reaction to generate chain termination extension products.


In addition to real time sequencing, the length of the sequence that may be analysed is in excess of 500 bp. The strands are then separated by electrophoresis under denaturating conditions and analysed separately. Analysis of the reaction products by electrophoresis is as already de­scribed for Sanger method. Consequently, DNA repositories and DNA assembly standards need certain adaptations to facilitate engineering using plant chassis. Gbparts are the minimal standard building blocks and they are classified in different categories according to their specific function. Promoter’s (PROM) prefix is GGAG and its suffix is AATG, which is the same as the coding region’s (CDS) prefix. Internal Type IIs recognition sites (exemplified here with the GGTCTC BsaI recognition site) are mutagenized during domestication following a standard procedure. By using special GB destination vectors in the reaction, we make sure that the resulting TUs can be subsequently used to build multigene constructs (constructs comprising several TUs within the same destination plasmid).
All the DNA parts composing a basic structure (PROM, CDS and TER) are mixed together in one tube with a GB destination plasmid, BsaI and T4 ligase. Two TUs assembled in complementary ?-level plasmids are combined in a ?-level destination vector to create a two genes module. The ends are not actually sticky; rather, the term denotes that the overhang allows these fragments to bind more readily with other strands. Cleavage with A gives fragments 2 and 7 kilo bases from a 9 kb molecule, hence we can have position of single A site from one end. Unlike plasmids, PCR products are short and re-annealed rapidly, so preventing the re-annealing process or biasing the amplification towards one strand by using a primer ratio of 100:1 can overcome this problem to a certain extent. Automated direct PCR sequencing is increasingly being refined, allowing greater lengths of DNA to be analysed in one sequencing run.
Without letting aside BioBricks, we decided to use the GoldenBraid system (GB) to build several of the intermediate genetic constructs employed in this project.
The same happens with the CDS and the terminator (TER), which share the part identity overhang GCTT, the first one as its suffix and the second one as its prefix. GB destination vectors are T-plasmids, a special type of plasmids used for plant transformation. As result of the restriction-ligation reaction the correctly assembled transcriptional unit is obtained. Therefore, by using two restriction enzymes, we can be more certain that our DNA has been digested. GB is a DNA assembly system specially conceived to facilitate genetic engineering in Plant Synthetic Biology projects (visit gbcloning.org for more information).
Therefore the new TUs assembled in GB vectors can be directly transferred into plants using Agrobacterium-mediated plant transformation.



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