KEENAN+W.

=The Discovery Process of BL21 - Ampicillin Resistant Bacteria=

__**Introduction:**__ When researchers are trying to analyze a specific protein, there are many factors and questions that come up which determines how they will be able to obtain it. Depending on what protein you want, different methods and strategies will be tested, including the type of cell used to grow the protein (mammalian or bacterial cell). The process could take a few hours or it may expand over a period of several days or months. However, every protein is unique and different and finding a common method to express all proteins is implausible and unreasonable. There are research methods that could yield a substance, but it is questionable whether if it is the protein that you want and some even fail to express the desired protein altogether. In this experiment, we are interested in finding out about protein BL21(DE3) - ampicillin resistant bacteria within //E. coli//. Most importantly, we are also interested in learning various processes of obtaining and analyzing our protein in order for us to use the same methods and strategies for future expression of proteins. The purpose of the three step process of protein expression, purification and characterization done in the experiment was to cultivate and grow a species of bacteria to express a recombinant protein and extract the desired protein out of the cell for future analysis. With this renown technique, it is hypothesized that the aftermath of these several experiments would yield our over-expressed protein BL21. Whether or not how well we were able to extract/purify the protein from other substances depends on our accuracy and precision and the use of sterile techniques during our performance. The better we are able to follow these parameters will put a limiting cap on simple mistakes, minimizing sources of human error, and therefore producing more reliable results.

__**Materials & Methods:**__


 * Protein Expression**
 * 1) In the beginning step of our experiment, we will cultivate and grow our protein on an auger plate with ampicillin in order to see if the protein we want is really ampicillin resistant. A control group will be made in which bacteria without this strain of DNA will die while bacteria in our experimental group will survive because the ampcillin present on the plate will have no effect on our // E. coli // cells. Amp-resistant cells will grow purple in color due to an added stain/dye. With a tube of 50ul of competent bacterial cells ( // E. coli - // BL21(DE3)), pipette 25ul of bacteria into two transformation tube. One would represent our DNA tube and the other will be our control group. Spin down the transformation tubes of bacteria using a micro-centrifuge instrument. Add 1.1ul of plasmid DNA into the DNA tube only. The control should have no DNA. Heat shock the tubes using a hot water bath for 45 seconds (mainly to fuse the added DNA in with our bacterial cells) and place them in ice for 2 minutes. Add 200ul of SOC media into the tubes and place them in the shaking incubator of 37 degrees Celsius at about 250 rpm for 30 minutes.Pipette 50ul of the solutions into their corresponding auger plates and using 5-6 collirollers to disperse the solution. Place them back into the incubator and let bacteria grow overnight.
 * 2) In the following day, take out your plates. As expected, the control did not yield any bacterial growth, while our experimental group showed many purple colonies of bacteria. Using a sterile pipette tip, transfer a single colony of purple protein into two round-bottom tubes filled with 10ul of ampicillin and 5ml of LB. Place in the shaking incubator for 8 hours at 37 degrees Celsius at 200-350rpm.
 * 3) Now we need to over-express our protein in order to work with it. Add 25ml of fresh LB in two Erlenmeyer flasks and add 50ul of Amp stock to each flask. Transfer 625ul of the starter culture form the tubes to each flask. Place in the shaking incubator for 16-24 hours at 37 degrees Celsius at 200-350 rpm.
 * 4) The media should now be purple. Take a 500ul sample of the flask, place it in an Eppendorf tubes and label it Sample 1. Pour the rest in 50ml conical tubes and centrifuge them at 4 degrees Celsius at 5000 rpm using Allegra X-15 for 10 min. Purple pellets should separate from the solutions to the bottom of the tube. Empty the remaining liquid into the liquid waste container. Add 5ml of 1X PBS solution to the conical tubes with pellets. Add 100ul of lysozyme to the conical tubes and re-suspend the material using a vortex machine. Lysozyme helps break down the bacterial cell wall in which all of the cells content is released in the solution. Store the tubes in a -20 degree Celsius freezer and clean up all your materials and lab station.


 * Protein Purification**
 * 1) Now that we have our over-expressed protein (BL21(DE3)), it is now time to extract it from debris and other waste that we do not want (the process of purification). The protein we want is in the purple pellet along with other material from the bacterial cell. Thaw the conical tubes by placing them in a beaker of water. Add 2ul of Cyanase/Benonase into the 50ml conical tube and invert it 5 times and incubate for 15 minutes. Cyanase digests the DNA and RNA in the solution and makes it less viscous.
 * 2) Distribute the lysate into two 1.7ml micro-centrifuge tubes and centrifuge for 20 minutes at 4 degrees Celsius at 14000 rpm. This divides the solution into two parts, a soluble fraction (liquid) and a debris pellet. Take a 50ul sample of the supernatent and label it Sample 2.
 * 3) Transfer the liquid supernatent (soluble fraction) into a clean 15ml conical tube leaving the debris pellet behind. Prepare the following buffers: Wash buffer (90ml of final concentration 1X PBS with 20mM imidazole) and an Elution buffer (10ml of final concentration 1X PBS with 250mM imidazole). Dispense the buffers into their corresponding 15ml conical tubes and mix.
 * 4) Filter the lysate though a 0.45um syringe (PES) with a 5ml syringe into a 14ml round-bottom tube. This will get rid of large particulate matter but not our protein.
 * 5) Now it's time to go through the purification process though a combination of batch and column chromatography. Add 0.5ml of Ni-NTA resin into the tube and mix well. Set up a Bio-Red Chromatography Econo column and add your lysate with resin and buffer into the column. Wait a brief moment for the material to settle. Now perform a series of column runs (collecting the liquid from each run in a clean tube): Waste, Wash and Elution 1 and 2 using the buffers made before and taking samples of each (Samples 3-6). Store them in the 4 degree Celsius fridge with the other samples along with Elution 1 and 2 conical tubes.
 * 6) The nanodrop machine was used to measure the absorbency of our samples at the general wavelength of 280nm and at the maximal wavelength of 574nm. Clean up your lab station and prepare for the third and final process of the lab.


 * Protein Characterization**
 * 1) With all our samples collected throughout the labs, we need to test them to see where our protein lies. This will somewhat help us determine whether or not if our protein is purified by following the steps that it has been though. Prepare your samples 1-6 though heat block and centrifugation for 2 min at 5000 rpm. Then add a blue stain buffer to each sample. Obtain a precast gel from a mentor and load it into an electrophoresis module filling up the container with 1X TGS buffer. Dock 20ul of each sample into a corresponding well (the first being your molecular standard as a guide). Then close the container, set it to 200 V and run the gel for 25-45 minutes.
 * 2) Afterwards, remove the gel from its case and put it on a piece of Whatman filter paper and wrap it with saran wrap or cellophane. Dry it using the Biotech lab drying machine. Set temperature to 75 degrees Celsius on gradient cycle for 1.5 hours. Turn on the vacuum and start the cycle.
 * 3) There should be lanes with blue streaks where there were docks of our samples. The various bands in the lanes represent a different type of protein (due to their molecular weight and properties). Clean up your lab station.

__**Protein Lab Results:**__
 * **Protein Expression**
 * **Protein Purification**
 * **Protein Characterization**

(Right) Auger plate with BL21(DE3) protein and ampicillin. Amp-resistant bacterial colonies grew and are stained purple (counted to be about 400+ colonies). (Left) Auger plate (Fun Plate) used to collect bacteria from a dirty sponge in the Lab. Bacteria is present.
 * Figure 1.** (Top) Auger plate has no BL21(DE3) protein with ampicillin (Control group). No bacterial growth.
 * Figure 2.** To save time, Neusha's auger plate with BL21(DE3) protein and ampicillin was used for the experiment instead of my own.



**Figure 3.** Flasks containing LB, ampicillin, BL21(DE3) and pGEM-gbr22, covered with foil after 18 hrs in shaking incubator.




 * Figure 4.** Centrifuge tubes after centrifugation. Purple pellet at the bottom of tubes contains bacteria BL21(DE3) with purple protein encoded by plasmid pGEM-gbr22. Wet pellet is kept while liquid is disposed. The weight of the wet pellet for Samples 1 and 2 are 0.45 and 0.35 grams respectively.


 * Figure 5.** Elutions 1 and 2. Elution 1 contains more of our bacterial protein while Elution 2 contains less of our protein.


 * Figure 6.** Trial 1 and 2 absorbance readings for Elution 1 by nanodrop spectrophotometer at 280nm. The absorbance reads to 0.465 and 0.450.


 * Figure 7.** Trial 1 and 2 absorbance readings for Elution 2 by nanodrop spectrophotometer at 280nm. The absorbance reads 0.199 and 0.182.


 * Figure 8.** Trial 1 and 2 absorbance readings for Elution 1 by nanodrop spectrophotometer at maximal wavelength of 574nm and 280nm (for comparison purposes). The absorbance readings at 574nm is 0.022 and 0.022.


 * Figure 9.** Trial 1 and 2 absorbance readings for Elution 2 by nanodrop spectrophotometer at maximal wavelength of 574nm and 280nm (for comparison purposes). The absorbance readings at 574nm is 0.005 and 0.009.


 * Figure 10.** Brief calculations for Beer's Law calculations (A=Ebc) estimating the concentration of our protein in Elution 1 and 2 at the wavelength of 280nm (left) and maximal wavelength 574nm (right).


 * Figure 11.** Denatured protein stained onto gel after electrophoresis (wet). Error: electrophoresis machine used was broken and only ran halfway.

** Figu **** re 12. ** Brianna's gel samples were used instead of my faulty gel. Her partner Anh did the first set while Brianna did her Wash, Elution 1 and Elution 2 samples. The one on the left is a wet gel version after electrophoresis and the one on the right is the dry version of the gel.

__**Discussion:**__

Only one of the plates showed signs of bacterial growth after the overnight incubation process. The plate that had the plasmid DNA showed bacterial growth (represented as purple colonies) while the control group did not. This is because the //E. coli// cells had a gene/strain for ampicillin resistance that was obtained though heat shocking of the cells. After incubating the transformation tubes for 8 hours, they had a foggy appearance. This indicated that our starter culture was successful and that we can proceed with producing/over-expressing our protein. After 18 hours, the larger cultures in the Erlenmeyer flasks appeared a pinkish purple color indicating protein cultivation success. The purple color representing our transformed bacterial cells expressing our protein BL21. After centrifugation, purple pellets were left on the bottom of the tubes of our solutions. It is made up of debris of the whole cell along with our protein. Lysozyme was added to the solution to break up the cell wall in order to release its contents. We now have a solution that we could use for the purification process. The biggest source of error in this process was contamination. Because we are working with live specimen, it is important that no other bacteria enters our culture sample. If this did happen, this could effect the growth of bacterial BL21 cells due to competition or even inhibit the expression of our protein. The other bacteria may compete with our //E. coli// cells resulting in the decline of BL21 cells survival rate. Although contamination may have occured, it is of minimal amount because our bacteria grew to maturity and we were able to express and cultivate our protein.
 * Protein Expression**

In this lab, we used cyanase to digest our DNA and RNA while lysozyme digested the bacterial cell wall releasing DNA and its contents. We then centrifuged the sample and took out a clear soluble fraction which contained our protein. The debris this time contained only the substance from the cell that we do not want and therefore it was discarded. Several runs of our solution though the column helped us purify our protein. Ni-NTA resin was added to our solution so that our protein can bind to it making it difficult for it to leave the column while debris and other substances were drained though. Histadine 6 tags, also present within the solution, binded to the resin as well further inhibiting our proteins from flowing though the column. A Wash buffer run was performed to get rid of hanging and loose particles that still may be attached onto our resin while an Elution buffer was suppose to release our protein from the resin. As more imidazole was added to the column our proteins were able to get released from the bonds of the resin and flow out into our elution tubes. This is because imidazole is a better binding substrate that attaches to nickle. It acts as a better competitor than our protein and therefore, takes the place where our protein originally was. In other words, the large amount of imidazole competed with the histadine 6 tag eluting the proteins off of their bonds. There was significantly large amount of protein located in Elution 1 than Elution 2. The darker shade of purple is an observation that explains this other than the graphs. Absorbency measurements are higher for Elution 1 than Elution 2 at the general wavelength of 280nm and at the maximal wavelength of 574nm.Therefore, Elution 1 contains more of our protein than Elution 2. In other words, Elution 1 has a higher concentration of our protein of 1.58mg while Elution 2 only contains 0.635mg of our protein. Some potential sources of errors would include whether or not our instuments were and techniques were sterile. The use of a contaminated instrument or careless techniques could introduce a new and foreign bacteria that could effect the outcome of our protein readings. When we tried to pipette the soluble fraction out, the debris could have been disturbed and could be transferred which could effect the outcome of our measurements and the way it reacted to the Ni-NTA resin. Some of our protein could of been denatured due to some time outside the ice bath which could affect the outcome of concentration readings. Lastly, there is always the question of whether or not our measurements were precise and accurate. A flaw within our measurements could contribute to a change in volumes for our samples.
 * Protein Purification**

The protein characterization lab allowed us to use the electophoresis module in order for us to analyze samples prepared in previous labs (Sample 1: the protein is located in the wet purple pellet along with other debris from the bacterial cell. Sample 2: the protein is located in the clear soluble fraction of the supernatent. Sample 3: the protein is located in the column binded to Ni-NTA resin and histadine 6 tag. Sample 4: the wash sample contains imidazole, debris and maybe some of our protein. Sample 5: enough imidazole was added to get rid of most of our protein. Sample 6: another run of imidazole to get what is left of our protein in the column). This could also be a lab where we are determining how "good" our results were from previous labs. Figure 12 was used as a basis for analysis of the data. Samples 1 - 4 (Wells 2- 5) had many bands present which suggest that were other miscellaneous debris or foreign proteins that has been present with our proteins (samples 1 & 2 ). It could also mean that our protein was in the process of purification there was a lot of collected debris/ foreign protein gathered from the purified protein solution (sample 3 & 4). However, because there are so many separate bands, we do not know whether or not if some of our protein escaped from the column when it shouldn't have. Samples 5 & 6 are our elution samples and contain our proteins and nothing else. We should see only one band in these lanes. This was somewhat successful. Sample 5 showed a blurred band which means that our protein was not a 100% purified. However, I like to say that it was about 60% purified. From this data we used a molecular weight standard to figure out the molecular weight of our protein in Kilodaltons (kDa). I decided that it was around 24-26 kDa. As it turns out the protein did measure out to be about 25.8 kDa from protein purification lab. Nonetheless, we can't be too sure if this is our protein. A Western Bolt or Protein Mass Liquid Chromatography can be used to verify our collected protein. A big source of error would be when we were trying to place our samples into the wells. Our samples could have easily spread and could have contaminated our other wells which could lead to extra bands that represent other contaminants and not our protein. Something called "smiling" of the gel occurs when solutions on the outside curve inwards and contaminate the readings of the other wells. The electrophoresis module was a big source of error. It was broken in the time of use and was not running properly. This could hinder the process of lab work and can even put a permanent stop to the experiment. The use of non-sterile equipment can introduce new bacteria and contaminate our results leading to unreliable data.
 * Protein Characterization**

__**Conclusions:**__

In this lab, we were able to successfully create a culture of BL21 bacterial cells and have them transformed with the plasmid DNA with the help of the process of heat shocking (our vector). Adding lysozyme and centrifuging the substance/solution allowed us to over-express the protein in a purple pellet containing debris from the cell and our protein. This lab is important for future experiments of VDS because we learn how to express a specific protein in competent bacterial cells. This might be useful in order to gather a large amount of a desired protein for analysis.
 * Protein Expression**

In this lab, we were able to successfully purify our over-expressed protein from our first lab bacterial Protein Expression. We used Beer's Law to help us calculate the concentrations of our proteins given the experimental absorbency measurements. From the collected data, we can conclude that Elution 1 contains more of our protein than Elution 2. We could also conclude that as the concentration of our protein increases, the absorbance increases as well. The protein collected in Elutions 1 and 2 can be used in our next lab Protein Characterization for further analysis on its structure and specific functions. This type of purification technique will likely be used for future VDS targets as well.
 * Protein Purification**

In this lab, sodium dodecyl sulfate polycrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins present in samples 1-6. Our protein was denatured giving the ends separate charges that dictate the direction of flow from the wells down to the gel. Then our gel went though a drying process. Our gbr22 protein in sample 5 was able to be identified using a molecular weights standard indicator. After verifying the contents of our protein, it could be used to test out enzyme activity and therefore test possible drug inhibitors.
 * Protein Characterization**

__**References:**__

Institutet. K; Protein production and purification. //Nat Methods// **2008,** 5, (2), 135-46.
 * Journal** **Articles**

Smith, D. B.; Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. //Gene// **1988,** 67, (1), 31-40.

http://www.embl.de/pepcore/pepcore_services/index.html
 * Websites**

https://www.neb.com/applications/protein-expression-and-purification