Drew+L

__ Title __ The Epic Bacteria Protein Lab Trilogy

__ Introduction: __ Recombinant protein, has increased in use as technology has spread to the wider population of the public.[2] The process involves tweaking existent bacterial DNA, to reach a wanted protein strand. The DNA is often replicated via PCR, then expressed at a low temperature, and purified in a buffer solution. Similarly this lab involved the expression of the protein by growing the cells, the purification of them using buffers and a column and finally the characterization using gel electrophoresis. The objective of this lab was to properly use the techniques of expression, purification and characterization to get a proper read on the molecular weight of the original pGEM-gbr22. __ Materials & Methods: __ Bacteria was placed in transformation tubes, spun, plasmid added and iced. The tubes were then heat shocked for 45 seconds, iced for two minutes and SOC media added to them. Bacteria was added to the plates and collirollers added as well, the plates and the plates were inverted and stored in the incubator. Pictures were taken of the plates, and the bacteria from the ampicillan plate was placed in a flask of LB media. Fresh LB and ampicillan were added to the flask and the tube was placed in the incubator overnight. A picture of the sample was taken, and poured into conical tubes and centrifuged( for 10 minutes and 5,000 RPM). Liquid was poured out of the tubes, and the pellet was weighted. Then 1X PBS was added to the conical tube, as well as lysozyme and the tube was put away. Buffers were made, the protein was syringe filtered, and Ni-NTA was added. Then the protein mixture was put in a column, and the flow was labeled as waste which was ran again. Then some was collected for sample three, then immadazole in 1X PBS was ran through and some was collected as sample four. Then the Elution buffer was ran through ( 1X PBS and 250mM Imidazole) and kept in conical tubes as Elution one and two( pictures were taken, and more samples collected). Then using Elution one on the nanodrop, the maximal wavelength was recorded. 10 microliters of 6X buffer was added to samples 2-6. These samples were then added to gel, and the gel placed in the container then two militers of the TGS was added to each. Then each sample was added to an individual well, the red connected to red and black to black and ran for 25 minutes at 20 volts. The gel was then removed and stained and placed in a shaker for 1.5 hours. It was then destained and left overnight. A picture was taken of it, and it was then dried on a drying bed on gradient cycle at 75c for 1.5hours. After which the resulting gel was used to determine the MW of the protein. __ Results: __

Using Beer's law A=EBc. You get at 280 nm .283+.254= .2685 as an average A= Coefficient* concentration .2685=39100 *c 6.87 nL At 574 the coefficient is 118300 so .102+.09 /2 = .096 .096= 118300 c .81 nL

__ Discussion: __ The multiple lines in well 9( where my sample 5 was) shows that the protein was not pure. There could have been an error in the purification process, as I may not have retained the flow in the column enough, which could've messed up my results. Lysozyme was used to damage the cell wall of the bacteria, and make it easier to purify the protein. Benzonaze and cyanase are used because they are nuclease that helped lower the fluidity of the protein, by digesting the unwanted DNA and RNA. Sample one held the culture before the centrifuge, sample two contained the protein after the lysate, sample three was after purifying the protein with the waste, sample four was after purifying it with wash buffer, and sample five and six came from elution one and two respectively. The difference between the wash and elution buffer is that the elution buffer had much more imidazole then the wash buffer by over 10 times. [1]His tagging works by adding the Ni to the protein, and then purifying it, it is then tested later ( like we with the gel) for when the Ni is present or not. The estimate MW of my protein is 25 Kilodaltons. There were two bands of equal intensity and a third one, so my sample was 33-50% pure. __ Conclusions: __ This lab used common lab techniques, to purify and character the protein we used. This taught another technique to figure out the molecular weight, by using a protein ladder, to get the purity and weight of the tested protein. This applies to VDS, when in the fall and summer we get individual proteins to express and purify and characterize. These techniques will be useful in future labs, in which we need to purify a protein and figure out its concentration via the HIS tag system. __ References: __

1. Berrow, N. S.; Bussow, K., Recombinant protein expression and solubility screening in //Escherichia coli//: a comparative study. //Acta Cryst// **2006,** (D62), 1218-26. 2. Gräslund, S.; Nordlund, P., **Protein production and purification**. //National Methods// **2008,** //February// (2), 135-46.