The cloning of Atrolysin A from Crotalus atrox
We are going to clone the gene for the toxin Atrolysin A,Genbank accession number U01234. The toxin is produced by Crotalus atrox, which is the Western Diamondback. The E. coli that we will use as a vector will not be effected by the toxin if produced. The Atrolysin toxin affects platelet adhesion inside the body. The toxin works best at a pH of 8.0 and denatures in an acidic environment. This is how the snake can eat its prey and not be affected by the venom. The Atrolysin A is only found in mRNA sequence so we have to isolate the coding sequence and amplify it with PCR. Once we have the gene isolated we need to amplify it and see if the coding sequence grows from its initial 1260 base pairs. If it does this means introns are in the sequence. If introns are involved we then must remove them. Removing them by splicing and PCR. We have also discovered a restriction site in our gene and it has to be removed by site directed mutagenesis. If we are able to isolate and clone the coding sequence, we will then test the protein on SDS gels against a control E. coli to see if the protein is present. Our primers we are using are Forward: 5’ ATG GAA AGA CTC ACC AAA AGA TAT GTT GAC CTT GTC ATA GTT G 3’. The Reverse Primers: 5’ TCA AAT CTG AGA GAA GCC AGA GGT TGA TTT GTA GGC TGT A 3'
- first item Make DNA buffer and prepare for the DNA extraction from the snake skin. Buffers needed are Tris/HCl buffer and TE buffer.
- second item Use agarose gel electrophoresis to find if the DNA is present in our buffer solution.
- third item Once we know the DNA is present we must then PCR the sample to amplify the coding sequence.
- fourth item With the amplified sample we then will find our coding region and sequence it to see if the coding sequence grows from 1260 base pairs. After sequencing we then must look at the sequence to see if we can view what might be introns.
- fifth item If introns are present depending on how many are in the sequence we then must remove them with site directed mutagenesis
- sixth item Once the introns are removed we must then remove the Restriction site PstI with site directed mutagenesis
- seventh item Once we have all the introns and restriction site removed we must then add the biobrick parts and then insert the DNA into our vector. Our vector is BBa_I0100.
- eighth item Once then DNA is in our vector we must grow the E. coli then test with controls.
- ninth item Testing will be done using SDS PAGE. We will compare the length of the control and our experiment E. coli. If the length of the experiment is longer than the control, then the gene for Atrolysin A is present.
File:RDT final presentation.pptx
Media:RDT final presentation.ppt.pptx
"Agarose gel electrophoresis." OpenWetWare. October 2012. http://openwetware.org/wiki/Agarose_gel_electrophoresis.
Anderson, John. Part:BBa_I0500. 4 Aug. 2006. Registry of Standard Biological Parts. 4 Aug. 2006. http://partsregistry.org/wiki/index.php?title=Part:BBa_I0500.
"Crotalus atrox hemorrhagic toxin a, atrolysin a (Ht-a) mRNA, partial cds." National Center for Biotechnology Information. 2012. http://www.ncbi.nlm.nih.gov/nuccore/U01234.1.
Eguchi, Tomoko and Yukinori. High yield DNA extraction from the snake cast-off skin or bird feathers. 19 May 2000. University of Ryukyus. 19 May 2000. http://scholar.google.com/scholar?hl=en&q=getting+DNA+from+snake+skin&btnG=&as_sdt=1%2C16&as_sdtp.
Fetzner, James W. Extracting High-Quality DNA from Shed Reptile Skins: A Simplified Method. June 1999. Brigham Young University. June 1999. http://www.biotechniques.com/multimedia/archive/00014/99266bm09_14694a.pdf.
Site-Directed Mutagenesis. 22 Aug. 2012. Wikipedia. 22 Aug. 2012. http://en.wikipedia.org/wiki/Site-directed_mutagenesis.
"Standard PCR Setup." Openwetware. October 2012. http://openwetware.org/wiki/840:153g:Materials.
Important Results and Milestones
- keep track of your most important results and refer to the corresponding page in your notebook
- upload important pictures (don't forget to label them! Powerpoint is very convenient). Remember: these will become quite handy later in your summary report or final presentation. If you do label and upload the pictures as soon as you got them, your summary report can be written much more effortlessly (do you usually procrastinate? This is chance to do some work before hand that frees you up for finals week).