ARIEL+C

Comments:

Intro should be more background info on theory or on the technique than restating methods Include units in Beer's Law calculations for further clarity Improve references

__ Introduction: __
Proteins play some of the most crucial roles in biological processes, so they are naturally the subject of extensive scientific study. Certain types of research into proteins require the proteins to be isolated and separate from the living organisms they are typically found in, and this may be done through the processes of protein expression, purification, and characterization [1]. These proteins are typically created from recombinant DNA plasmids and used in a variety of fields of study, such as biomedical research [2]. The plasmid must be inserted into a host cell (some common hosts are bacteria and yeast), typically with heat shock. //E//. //coli,// and in particular the strain BL21(DE3), is commonly used expression host for protein expression because it is effective [2]. The recombinant plasmid inserted usually code for antibiotic resistance to be sure that transformation with the plasmid has happened, and N- or C- terminus affinity tags like the 6-histadine tag for effective purification in later steps [1].

After transformation with the plasmid, protein expression and purification follow [1]. The protein of interest can be extracted by lysing the bacterial cells and separating the tagged protein from the other proteins and cell debris. To ascertain the purity of the solution containing the protein, it is characterized through the process of gel electrophoresis [1]. This experiment will involve transformation of //E. coli // BL21(DE3) with a plasmid vector encoding for the purple coral protein pGEM-gbr22, overexpressing this protein in a large culture, and purification and characterization of the protein. The hypothesis states that the plasmid will be transformed, gbr22 expressed and a purple color evident. After purification and gel electrophoresis, Elution 1 will contain one clear band representative of the protein.

__ Materials & Methods: __
The protein pGEM-gbr22 was first expressed in // E. Coli //. // E. Coli //BL21 (DE3) was placed in two different transformation tubes. pGEM-gbr22 plasmid was added to one of the tubes and the two tubes were then heat shocked in a 42°C water bath for 45 seconds, and then had 200 µl of SOC media added to them. The tubes were then put in a 37°C shaking water bath for 30 minutes at 250 rpm. The bacteria was then plated on agar plates and stored in a 37°C incubator overnight. The following morning, a culture tube containing a single transformed bacterial colony, 10 µl of ampicillin, and 5 mL of LB was prepared and placed in a shaking incubator at 37°C at 300 rpm for 8 hours. A flask containing 25 mL LB media, 50 µL of ampicillin, and 625 µL of starter culture was placed in a shaking incubator at 37°C at 300 rpm for 20 hours. 500 µL of the culture was saved as Sample 1 (to be characterized later) and the rest was centrifuged for 10 minutes at 5000rpm at 4°C. The pellet was resuspended in 2.5 mL of 1x PBS and 50 µL of lysozyme, and was stored in a -20°C freezer.

Next, the protein was purified. After the cell suspension was thawed, 2 µL of Benzonase was added, and the tube with the lysate was microcentrifuged for 20 minutes at 14000 rpm at 4°C. 50 µL of the supernatant was saved as Sample 2 and the rest was filtered through a syringe filter. Then, 0.5 mL Ni-NTA was added. The mixture was poured in to a chromatography column, then Wash Buffer, then Elution Buffer twice. 50 µL of flow-through, wash, and elution were collected after each step as Samples 3-6. The Nanodrop spectrophotometer was used to measure absorbance of the purified protein at both 280 nm and 574 nm in order to calculate the yield.

Sample 1 was microcentrifuged for 5 minutes at 5000 rpm and then the resulting pellet was resuspended in 200 µL of water. 40 µL of blue loading buffer was added. Samples 2-6 had 10 µL of 6x loading buffer added and properly mixed in. All samples were then put on the heat block at 95°C for 5 minutes and centrifuged for 2 minutes at 5000 rpm. Molecular weight standards were loaded into wells of the SDS-PAGE gel along with the various samples. The SDS-PAGE gel was run for 25 minutes at 200V. The gel was then removed, stained with Imperial protein stain, destained overnight, and dried on Whatman filter paper covered with cellophane on a drying bed for 75°C for 1.5 hrs.

__ Results: __






















No, I don't think I beat the other groups in getting the most bacteria

A= εbc MW of gbr22= 25794.2 g/mol <span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">ε= 38850 m-1cm-1
 * <span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">Determining protein yield: **

<span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">0.479=38850(1) * c <span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">c= 1.233 x 10^-5 mg/mL <span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">5mL * (1.233 x 10^-5)mg/mL= 6.165 x 10^-5 mg
 * <span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">At 280nm: **

<span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">10(0.842)=11830(1) * c <span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">c=7.117x 10^-5 mg/mL <span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">5mL * (7.117x 10^-5)mg/mL = 3.585x10^-4 mg
 * <span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">At 574nm: **

__ Discussion: __
In the expression portion of the lab, the plasmids for pGEM-gbr22 were shown to have been transformed into the bacteria successfully due to the growth on the ampicillin plates. After letting the cultures grow up some, the protein was evidently being expressed because a purple color was seen. After purification, collecting our samples (The six samples we collected were: 1- Cell debris, 2- Soluble components, 3- Flow through, 4- Wash, 5- Elution 1, and 6- Elution 2) and running gel electrophoresis, with reference to the Molecular Weight standard ladder, it seems that the target protein is around 25 kDa, which is generally consistent with the molecular weight of the protein found from literature online, which was 25,794.2 g/mol. However, the purity of the sample is estimated to only be around 40%, as two other bands, one of comparable intensity and the other with considerably less, can be seen in the lane with Elution 1, which should only have contained the desired gbr22 protein.

Potential sources of error in this lab include non-sterile technique and contamination. Contamination can occur at a variety of different steps, including at the very beginning. Because the HIS tag system works through encoding a 6-histadine tag at the terminus of the protein, having that tag bind to nickel resin attached to a bead to prevent it washing through, and finally being outcompeted by the Imidazole and flowing through as the elutions, it is difficult to imagine that other proteins would have resisted purification and flowed into the elutions as well if they didn’t have the HIS tag as well. So the contamination may have been with the plasmids. It is possible that another plasmid with the HIS tag and Amp resistance that would express a different gene had been in the same mixture as our pGEM-gbr22 plasmid.

Lysozyme is used in the lab after the proteins have been overexpressed in the bacteria, and they break open the cells so that after centrifugation, the cell debris can be separated from the water soluble proteins. Benzonase is added in a step after that in order to digest all the genetic material to leave the proteins.

The Wash and Elution buffers were different in that the Elution buffer had a far higher concentration of Imidazole—enough to outcompete the histadine tags on the proteins. The Wash buffer’s concentration of Imidazole was too low to release the HIS tags from the resin.

__ Conclusions: __
<span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">In the lab, the coral protein gbr22 that was overexpressed in //E. coli// was first <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">purified by lysing the cells, removing insoluble debris and with an affinity tag and Ni-NTA resin. Then it was characterized with SDS-PAGE gel electrophoresis analysis. The final resulting protein had a molecular weight of approximately 26 kDa and its purity was about 40%.

<span style="background-color: #ffffff; color: #000000; font-family: Arial,sans-serif; font-size: 13.3333px; text-align: start;">Future directions would likely include purifying gbr22 further to improve upon the 40% purity. <span style="background-color: #ffffff; color: #000000; font-family: arial,helvetica,sans-serif; font-size: 13px; text-align: start;">Expression, purification, and characterization are very likely to be used in drug screening to isolate a specific protein in order for various ligands to then be tested against the protein to determine how well various ligands inhibit the target proteins.

__ References: __
1. Mattaj, I. W. Protein Expression and Purification Core Facility. [] (accessed April 17). 2. Structural Genomics Consortium. Protein production and purification. Nat Methods. 2008;5:135–146. et al. 3. Thermo Scientific. PageRuler Prestained Protein Ladder #26616. [] (accessed April 11).