Analysis Of Gfp-Tagging

Submitted By angelabaydoun
Words: 712
Pages: 3

GFP-tagging Summary

Invented merely 30 years ago, the polymerase chain reaction, a technique used to create substantial amounts of specific DNA sequences in significantly short amounts of time, is even more so frequently used today in many biologically interdisciplinary fields. In the article, “Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins,” five researchers sought to further study microtubule dynamics, particularly in vivo, and more specifically, microtubule-associated proteins (MAPs). Microtubules themselves are prominent components of any cell’s cytoskeleton For these purposes, a modified version of the infamous green fluorescent protein was engineered, since the rate at which wild type GFP’s fluorophore is not naturally fast enough. Through PCR, the researchers were able to develop two expression vectors, pBact-NGFP and pBact-CGFP, in order to study these microtubule-associated proteins without damaging them. First and foremost, pBact-NGFP was constructed, as its formation is the precursor to the formation of pBact-CGFP. From a previously engineered pUHD 10.3, provided to the researchers, the coding for its gfp gene was amplified via mutagenic PCR. Subsequently, through the use of primers, 5’ and 3’ HindIII sites were created; 3’-primer was used to add sites for the restriction enzymes SpeI and EcoRV. All of this was then cloned into the existing HindIII site of a separate pBact 16 vector, creating a whole new vector. At this point, the mutant GFP S65T is introduced to this new vector via site-directed mutagenesis; the source of it is the vector pGFP-N1. The mutation lied within the NcoI-BsrI fragment on pGFP-N1, which then takes the place of the analogous NcoI-BsrI fragment on the new vector. This finally yielded one of our two expression vectors—pBact-NGFP. The entire gfp gene in their newly engineered pBact-NGFP was then to be introduced to another pBact 16 vector to yield the second expression vector. As before, mutagenic PCR was implemented to amplify the gfp gene and introduce HindIII restriction sites after the beta-actin promoter. However, 5’-primers were employed this time, and added sites for the restriction enzymes ClaI and EcoRV (Figure 1B), rather than SpeI and EcoRV. Lastly, this fragment was bonded to the aforementioned pBact 16 at its HindIII site and yielded the pBact-CGFP expression vector. All in all, it differs from the pBact-NGFP it that they have two restriction sites different from each other, and that GFP can be tagged on the C terminus, not the N terminus. After the construction of these vectors, MAP2c and Tau34, two microtubule-associated proteins, were fluorescently tagged with the GFP through PCR with the help of the pBact-NGFP; a 5’ SpeI site and a 3’ EcoRV site was created with primers. An 11-mer c-myc epitope peptide was added to the N terminus of the respective MAP2cs and Tau34s. These MAPs were able to bind to microtubules and cause a rearrangement in non-neuronal cells.