G. tigrina Hox gene DthoxC insertion into prokaryote E.coli, by UNIamCloning
DthoxC is one of several Hox genes, which make up the homeobox domain, which encodes for a homeodomain protein. The DthoxC gene regulates expression along the anteroposterior axis during bilateral regeneration in the planarian Dugesia (Griardia) trigrina. The Hox gene’s functionality is not testable in E.Coli, so an SDS- PAGE will need to be performed at the end of experimentation to show whether the transfer and cloning of DthoxC was a success. The DNA nucleotide sequence as well as the sequence for the amino acid translation product were located using the NCBI gene bank: accession X95416.1. The DNA sequence consists of a total of 942 base pairs and contains no introns. To clone the DthoxC gene, DNA will be extracted from Dugesia trigrina and a purification will be performed to clean up the sample. Then the gene will be amplified via PCR using flanking primers specially designed for the DthoxC sequence. The initial set of primers will be constructed using BioBrick-compatible ends. If these fail, a second set will be created without the BioBrick extensions. An additional two sets of oligos will be constructed as well, in order to compensate for EcoR1 sites within the gene sequence. Although three enzyme-problematic sites exist, two are within 18 base pairs of each other and can be fixed with two nucleotide alterations in one oligo. The segments sliced apart with the primers will either be ligased back together or amplified multiple times via PCR. Once successfully amplified, the gene fragments and vectors will be digested using BioBrick enzymes and the fragments will be spliced directly into the vector. Protein expression vectors will be used for this cloning process because Hox gene functionality cannot be tested for in bacteria and protein expression will be required for testing the effectiveness of the cloning process. The plasmid vectors, with newly spliced DthoxC gene fragments, will be transformed into chemically competent E.Coli bacteria. The bacteria will be grown on plates, after which the plasmid DNA will be isolated and sequenced to test for the presence of DthoxC gene fragments. Once determined that the DthoxC gene has been inserted successfully, it will be allowed to undergo transcription and translation, and with the addition of a strong promoter, its expression will be tested via SDS-PAGE, or sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Primary research article: [http://dev.biologists.org/content/124/1/ 141.full.pdf+html]