Analysis of the DBR Gene
We describe the techniques behind the recombining process that enables us to insert a modified affinity tag with neomycin resistance known as “TAP-neo” in between the Gal and Z fragments of the RABBI gene. Similar research has shown evidence of successful recombination events [3,5 and 6] that have ultimately led to purification procedures of the affinity tagged fusion protein complexes which permit mass spectrophotometers analysis [1 4,5,6,7 and 8].
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Optimization of the earlier steps within this experiment that serve as preliminary procedures needed to yield a reified plasmid product from the page-T Easy (Promote) vector are described in detail. Future testing is needed before beginning recombining, selection of homologous recombination and purification of the TAP-tagged RABBI protein product. Introduction The RNA-binding motif protein 22 (RABBI) has known interactive functions involved in the early stages of pre-morn splicing activity [2,3] as well as nuclear transaction of certain calcium-binding proteins .
It contains a conserved RNA-binding domain, a zinc finger [2,3] uniquely downstream of a C-terminus rich in the amino acids Glycerin and Praline . Found in both the nucleus and cytoplasm of eukaryote and yeast [2,3], RABBI binds directly to the internal stem-loop (SSL) domain of teeth asana and to the pre-morn intro near the 5′ splice site . Actively involved with the splice’s and its early functions [1 in the cell, further observations have as it moves from the costly to the nucleus.
As part of the SLIT 1 family, which contains a short group of highly conserved eukaryotic proteins associated with speleological activity [INCUBI, 2012], RABBI is similarly composed of a variety of structures enhancing its ability to recognize and bind to both asana and pre-morn. Behind this experiment, through a process known as recombination-mediated genetic engineering (recombining) , we are attempting to isolate this protein in large quantities through affinity tagging in order to further understand its unique role in the cells of eukaryote and yeast.
Assembling DNA fragments with this tandem affinity purification (TAP) [1 and 8] sequence involves inserting a “tag” between the 2 “Z” fragments comprising the gene of interest . Recombining is a method that utilizes a group of 3 expressed proteins from bacteriologic lambda known as game, bet, and ex. Together, they work to alter a DNA product derived from PC by making its terminal ends single stranded and 8] while subsequently enabling homologous recombination into a bacterial plasmid vector [4,5,6 and 7].
Subjoining of this product into the blueprints II KS(+) (Agilest) is followed by sequence elimination and insertion into a stock of 4TH cells purified from 5 x 106 cells using the Generate Mammalian Genomic DNA Nipper Kit (Sigma-Aldrich) via electrification. Isolation of cells with the encoded RABBI gene is made possible by inserting an additional gene coding for neomycin resistance [2,4]; this was done prior to subjoining. Upon introduction of 6418, an macroeconomics antibiotic, only cells with this gene being expressed will survive .
Following purification, RABBI and its associated proteins form a native complex that can thereby be prototypically digested  which allow for individual peptide fragments to be analyzed by mass spectroscopy. The evolutionary distance between human and chicken will also allow us to assess the evolutionary conservation of the protein machinery . Additional information may potentially contribute to our understanding of the exact distinct functions of the RABBI protein. Results
Prior to reaching the recombining portion of this experiment where it becomes possible to fuse the TAP tag in between the Z fragments of the RABBI protein coding gene, a series of changes in the earlier steps were required. Multiple alterations were made within these preliminary reactions in order to optimize experimental conditions. The initial PC program contained a changing heat interval as follows: xx (coccyx 1 5″, coccyx 30″, coccyx 2:30). The resulting product from this program contained an average concentration of DNA (Figure 3). In order to derive a larger yield, the interval of repeat cycles was increased from 30 to 35.
This alteration in programming produced no apparent amplified DNA fragment (Figure 4). A final change reverted back to the original xx heating interval but raised the annealing temperature from DNA product (Figure 5). Following purification and ethanol precipitation of the amplified RABBI-Z gene fragment, a PC product concentration of 61. Eng/Pl was produced. 83. Eng of purified PC product was introduced into a legation mix containing Eng of the page-T easy cloning vector (Promote) and 3 units oft DNA Aliases (Promote). Following incubation, Pl of this reaction was inserted into an aliquot of E.
Coli XSL-II Gold Cells prepared using the Z competent E. Coli Transformation Kit (Zoom Research). After 24 hours of uninterrupted cell growth at ICC, 19 white colonies were produced indicating insertion of the RABBI-Z gene fragment. Following purification using the Ferment’s Genet Plasmid Nipper Kit, the isolated plasmid DNA underwent analytical restriction digestion. The procedure used the purified DNA of 6 different colonies from the chemical transformation, each within a Pl sample along with 10 units of the Not’ (New England Bolas) restriction enzyme.
Following gel electrophoresis, the digestion reaction showed little to no proper insertion of the gene product into the page-T easy (Promote) vector (Figure 7). Due to the high volume of repeat trials needed to properly progress through the experiment, digestion was the furthest point reached. Resuming testing will hopefully allow for recombining and the eventual isolation and purification of the expected tagged protein product. Discussion The individual products of each experimental step within this entire process were useful for a variety of reasons.
As multiple different analytical procedures proved, here are sensitivities within the RABBI-Z gene fragment. These must be taken into account in order to successfully proceed towards recombination and any potential attempt at synthesizing and purifying the affinity tagged proteins. First, specifying a particular PC program that correctly amplified the gene was a lingering complication [Figures 3, 4, and 5]. A variety of alterations showed that the most efficient number of repeated heating cycles was 30 and that the preferred annealing temperature was ICC [Figure 5].
Another variable playing a large role in the success of the PC was the presence of DMS. After IV analysis, it was concluded that a reaction containing 6% DMS produced the highest quantities of amplified gene product [Figure 3]. Furthermore, original protocols called for the production of 2 equal PC reactions [Figure 3], this amplified the RABBI fragment to proper concentrations but lacked sufficient volumes to compensate for lost DNA. It was concluded that because an undetermined amount of DNA is lost over the course of multiple procedures, generating 4 PC reactions was preferred over 2 [Figures 4 and to exceed volumetric limits.
This was a result of incised agrees/DNA samples weighing an average of 0. 0 grams; this was twice the recommended maximum. Therefore, 2 unique reactions following gel incision had to be developed (to avoid overflow) which serves as a potential reason for how noticeable amounts of gene product could be potentially left behind between each subsequent procedure. A difficulty that arose later in the experiment was the designation of colonies that had properly cloned plasmid DNA with a confirmed gene insert.
After a 24 hour incubation period, certain colonies exhibited an off-white coloration with arguable tints of blue dispersed throughout. Even after 48 hours of incubation at ICC, the operation in color was not entirely definitive; such an ambiguity leads to a difficult inoculation selection process. It is likely that many colonies chosen for digestion contained little to no insert present and subsequently lead to the multiple failed procedures that followed. Due to the lack of a proper digestion, sequencing of the page-T easy vector for a proper insert could not be attained.