Luis+V.+(RP+Summ+14)

Week 14-15
Thank you for updating Luis. Excited about crystals! - Dr. B 12/171/14

__**Production of High Concentration and Pure BpACR for Crystallography**__ One of the goals of this research is to obtain a crystal structure of BpACR with the natural substrates NADPH and AAC docked, as well as the crystal structure of the enzyme with an inhibitor if one is found. To do this, a high concentration and pure protein product is needed. To do this expression was conducted in 2L of LB media, yet still concentrated to a very small amount in order to obtain the very high concentration needed.

After FPLC tubes 32-39 were obtained. Each tube contained 1.3mL of BpACR eluted in sample which means a total of 10.4 mL of sample were obtained. This sample was nanodropped to find the concentration.
 * __Reconcentration and Storage of FPLC__ **
 * Figure 1. ** Nanodrop of BpACR in FPLC buffer after reconcentration, samples 1 and 2.



Elutions 1 and 2 were run though the G75 Superdex column to further purify BpACR. The following was the result from the FPLC machine.
 * __FPLC__ **
 * Figure 1. ** Graph of FPLC of BpACR results. Tubes 32-39 were obtained for reconcentration.

A PAGE gel was made using samples obtained through purification. 1 - ColorPlus Protein Ladder 3 - Cell Lysate after induction (before induction sample was lost) 3 - Soluble Fraction 4 - Flow Through 5 - Wash 6 - Elution 1 (2.35 mg/mL) 7 - Elution 2 (0.53 mg/mL) There was some contamination on the Elution 1 and Elution 2 yields so we proceeded to do FPLC on these elutions.
 * __Characterization Using PAGE__ **
 * Figure 1. ** PAGE gel of BpACR using the following samples.

__ **Purification of BpACR using Nickel Chromatography** __ After the BL21(DE3) cells were centrifuged, sonicated, and had the soluble fraction extracted from them they were run through a nickel column to purify the BpACR. From this we obtained 5mL of Elution 1 with concentration of 7.71mg/mL and 10mL of Elution 2 with a concentration of 1.70mg/mL.
 * Figures 1&2. ** Nanodrop concentrations of Elution 1, trial 1 and trial 2


 * Figures 3&4. ** Nanodrop concentrations of Elution 2, trial 1 and trial 2

__ **Expression of BpACR in BL21(DE3) cells** __ To express the proteins the cells were grown overnight in the incubator. They were then transferred to 2 separate liters of LB and their absorbance was monitored every 30 minutes to ensure that they have grown enough before we induced expression. Once the absorbance reached .5 at 600nm IPTG was added to induce expression of BpACR.
 * F **** igure 1. ** Log of times at which absorbances were recorded and when IPTG was added.

Week 11-13
The first 3 compounds were purchased for inhibition assays. They were assigned the following labels: A - 7533374 (ranked 7th) B - 7613090 (ranked 17th) C - 6517271 (ranked 18th)
 * __A stab at Inhibition Assays__**

They were all dissolved in 100% DMSO to a final concentration of 50mM. Wanted to try an inhibition assay of compound A. To do this, the exact same recipe for enzyme assays was used, but right after the EAA was added compound A was added to a final concentration of 0.15mM.


 * Assay 20:**
 * Figure 8.** The compound did not appear to inhibit the enzyme activity any. Once I discussed the inhibition assay with Oscar I realized how many things I was missing from this assay. I should be running a control with DMSO only to determine if DMSO is possibly acting as an inhibitor or possibly even an enhancer of the enzyme's activity. I should also be running the assay at higher concentrations so that I can establish off the bat if the compound is capable of inhibition. The compound should also be incubated with the enzyme for several minutes before its addition to the mixture.

I want to see if I could use ethyl acetoacetate (EAA) as a substitute for AAC. This would greatly reduce the cost of assays since concentrated EAA can be bought by the liter - it is very cheap.
 * __Using ethyl acetoacetate as a substitute for acetoacetyl-CoA__**


 * Assay 19:**
 * Figure 7.** Enzyme activation assay of Gly2 using EAA as a substrate. Here the substrate clearly worked and it worked very well. The good thing about this substrate is that it gives a very straight line and since there is so much it does not run out before the NADPH runs out. This means we can record the slope for longer periods of time and get more consistent results.


 * Assay 18:**
 * Figure 6.** Enzyme assay of Gly2 using EAA as a substrate. This was quite obviously a failure. Too much EAA was used, creating an emulsion in the cuvette that was too hard to measure the absorbance through. We will try to fix this with less substrate.


 * Assay 17:**
 * Figure 5.** Enzyme assay of Gly2 using the usual AAC as a substrate. This was done to check and see if the enzyme was still working just fine, and it was.

At this point I had expressed twice. I wanted to check and see which protein was functional, and later I can look and the slopes and determine if there was any difference in reaction rate depending on how long the protein had been stored and if the storage method (glycerol vs. snap frozen). Will refer to the 4 different storages for my protein the following way. SF1 - Snap frozen 1, made July 2014. 64.76 uM SF2 - Snap frozen 2, made Sept 2014, 44.24 uM Gly1 - Glycerol 1, made July 2014, 51.808 uM Gly2 - Glycerol 2, made Sept 2014, 35.392 uM
 * __Checking to see what protein works__**


 * Assay 16:**
 * Figure 4.** Enzyme activation assay for Gly2. This assay shows that the Gly2 protein does work.


 * Assay 15:**
 * Figure 3.** Activation assay for SF2. This one went along as the assay normally does. The assay shows that the enzyme does indeed work.


 * Assay 14:**
 * Figure** **2.** Enzyme assay run using SF1 protein. Too much enzyme was added on accident, leading to the slope being very steep and tailing off at the very end. This protein is also functional.


 * Assay 13:**
 * Figure 1.** Assay for the Gly1 protein. The slope begins one BpACR was added. It seems there was some sort of malfunction, not enough AAC was added possibly. The slope shows that the protein is functional, however.

Week 9-10
__**ChemBridge30k Library**__ The chembridge 50k diversity library was partially screened against 3GK3. Only the first 30k compounds were screened. This was run on 20 processors. The results were the following. This is a list of the top ligands from the second run of virtual screening. Their properties are listed below. The top 5 compounds that fit several criteria will be highlighted yellow for possible ordering. These criteria include most importantly high gold score and low LogP(less than 3.25), but also other Lipinski properties.

**ID** ||
 * **Rank** || **Score (Run2)** || **Score (Run1)** || **LogP** || **# Hbond (Don/Acc)** || **Molecular Weight** || **Follows Lipinski?** || **ChemBridge**
 * 1 || 87.52 || 84.60 || 4.65 || 1/4 || 494 || yes || 6641100 ||
 * 2 || 86.30 || 63.69 || 4.63 || 2/4 || 487 || yes || 7633869 ||
 * 3 || 84.55 || 72.46 || 5.17 || 2/2 || 447 || No || 5109349 ||
 * 4 || 83.42 || 69.66 || 2.16 || 0/6 || 427 || Yes || 6061445 ||
 * 5 || 82.95 || 79.74 || 5.39 || 1/3 || 466 || No || 6642286 ||
 * 6 || 82.07 || 67.82 || 4.80 || 1/3 || 480 || Yes || 7638934 ||
 * 7 || 81.84 || 75.05 || 0.51 || 1/5 || 490 || Yes || 7533374 ||
 * 8 || 81.75 || 73.99 || 5.43 || 3/1 || 485 || No || 5159216 ||
 * 9 || 81.73 || 70.85 || 3.87 || 2/5 || 494 || Yes || 6255650 ||
 * 10 || 81.67 || 65.11 || 4.25 || 0/6 || 439 || Yes || 7007267 ||
 * 11 || 81.61 || 67.86 || 2.60 || 2/4 || 416 || Yes || 6541234 ||
 * 12 || 81.52 || 67.83 || 5.50 || 2/4 || 451 || No || 5194734 ||
 * 13 || 81.42 || 75.36 || 4.31 || 1/3 || 458 || Yes || 7507861 ||
 * 14 || 81.24 || 75.15 || 4.50 || 1/4 || 472 || Yes || 5187026 ||
 * 15 || 81.20 || 78.55 || 3.54 || 0/5 || 457 || Yes || 7012771 ||
 * 16 || 81.15 || 64.26 || 3.90 || 1/5 || 490 || Yes || 7630172 ||
 * 17 || 80.97 || 70.21 || 3.14 || 0/4 || 429 || Yes || 7613090 ||
 * 18 || 80.93 || 72.61 || 3.25 || 2/5 || 411 || Yes || 6517271 ||

The highlighted compounds are compounds that can possibly be ordered. Their structure and binding character is shown below.


 * **ChemBridge ID** || **2D Structure** || **Docking Image** || **Polar Contacts** || **Price Group** ||
 * 6061445 || [[image:20.jpg]] || [[image:25.jpg width="293" height="278" align="center"]] || 1 || 4 ||
 * 7533374 || [[image:19.jpg align="center"]] || [[image:24.jpg width="284" height="270" align="center"]] || 1 || 1 ||
 * 6541234 || [[image:18.jpg align="center"]] || [[image:23.jpg width="284" height="269" align="center"]] || 2 || 4 ||
 * 7613090 || [[image:17.jpg align="center"]] || [[image:22.jpg width="285" height="271" align="center"]] || 1 || 3 ||
 * 6517271 || [[image:16.jpg align="center"]] || [[image:21.jpg width="284" height="270" align="center"]] || 3 || 2 ||

__**Control Ligand Library**__ The following ligands were chosen for a controlled docking. The positive ligands came from the natural substrate, the natural substrate without CoA, CoA without the substrate, and bindingdb and journal articles.

**Positive** Acetoacteyl-CoA (only found 2d sdf) 3-hydroxybutyryl-coenzyme A (only found 2d sdf) (5,6-Diphenyl-pyridazin-3-yl)-octyl-amine from [] It was drawn on Marvin sketch and then pubchem was checked for a similar structure and redirected to [|https://pubchem.ncbi.nlm.nih.gov/compound/10316429?from=summary#section=3D-Conformer] 3-hydroxy-3-methylglutaryl-coenzyme A (only found 2D) alpha-aceto-alpha-hydroxybutyrate AN-698/42027016 (suggested by bindingdb) acetoacetic acid 3-Hydroxybutyric Acid Coenzyme A (only 2D) Acetyl Coenzyme A (only 2D)

**Negative** Aspirin 2-Butynedinitrile (Small) 5-methyl-5H-tetrazole (Small) CID15981164 (2D) CTK3H5722 (2D) CID15953394 (2D)

**Results**


 * Score || S(PLP) || S(hbond) || S(cho) || S(metal) || DE(clash) || DE(tors) || intcor || time || Ligand name ||
 * 90.84 || -71.09 || 6.25 || 1 || 0 || 0 || 4.49 || 3.94 || 218.877 || '3-hydroxybutyryl-CoA-2D' ||
 * 82.47 || -68.03 || 6.82 || 0 || 0 || 0 || 5.79 || 4.31 || 238.987 || '3-hydroxy-3-methylglutaryl-coenzymeA-2D' ||
 * 80.75 || -61.8 || 7.07 || 0.86 || 0 || 0 || 3.29 || 1.73 || 205.935 || CoenzymeA-2D' ||
 * 78.7 || -69.97 || 4.02 || 0 || 0 || 0.11 || 3.69 || 1.58 || 198.995 || 'CoenzymeA-2D' ||
 * 78.25 || -70.64 || 3.77 || 0 || 0 || 0.03 || 2.28 || 0.74 || 36.115 || 'AN-698-42027016' ||
 * 76.78 || -69.72 || 3.75 || 0 || 0 || 0 || 2.4 || 0.62 || 37.469 || 'AN-698-42027016' ||
 * 76.41 || -62.36 || 4.1 || 1 || 0 || 0 || 2.4 || 3.56 || 229.343 || '3-hydroxybutyryl-CoA-2D' ||
 * 76.26 || -63.59 || 6.59 || 0 || 0 || 0 || 5.86 || 4.63 || 221.636 || '3-hydroxybutyryl-CoA-2D' ||
 * 75.94 || -67.35 || 4.13 || 0 || 0 || 2.59 || 4 || 4.21 || 225.03 || '3-hydroxybutyryl-CoA-2D' ||
 * 75.77 || -59.58 || 7.35 || 0 || 0 || 0 || 4.72 || 3.59 || 239.736 || '3-hydroxy-3-methylglutaryl-coenzymeA-2D' ||
 * 75.52 || -64.28 || 5.38 || 0 || 0 || 0.35 || 4.28 || 4.01 || 212.273 || 'AcetylCoenzymeA-2D' ||
 * 75.09 || -50.9 || 9.28 || 0 || 0 || 0 || 2.59 || 1.54 || 174.501 || 'CTK3H5722-2D' ||
 * 74.71 || -58.49 || 5.9 || 0 || 0 || 0 || 4.11 || 4.38 || 244.037 || '3-hydroxy-3-methylglutaryl-coenzymeA-2D' ||
 * 74.61 || -68.24 || 3.06 || 0 || 0 || 0 || 2.86 || 2.91 || 222.061 || 'Acetoacetyl-CoA-2D' ||
 * 74.43 || -64.47 || 4.31 || 0 || 0 || 0 || 4.19 || 3.22 || 237.226 || '3-hydroxy-3-methylglutaryl-coenzymeA-2D' ||
 * 73.32 || -69.04 || 2.32 || 0 || 0 || 0 || 1.84 || 0.73 || 18.094 || 'AN-698-42027016' ||
 * 72.85 || -61.81 || 5.02 || 0 || 0 || 0 || 3.78 || 3.54 || 214.987 || 'Acetoacetyl-CoA-2D' ||
 * 72.77 || -60.89 || 3.06 || 1.98 || 0 || 0 || 3.63 || 3.99 || 209.942 || 'Acetoacetyl-CoA-2D' ||
 * 72.2 || -61.38 || 4.4 || 0 || 0 || 0 || 2.77 || 1.78 || 177.477 || 'CID15981164-2D' ||
 * 71.99 || -54.33 || 6.78 || 0 || 0 || 0 || 2.61 || 2.56 || 224.288 || '3-hydroxybutyryl-CoA-2D' ||
 * 71.71 || -59.23 || 4.55 || 0 || 0 || 0 || 2.92 || 4.67 || 225.654 || 3-hydroxybutyryl-CoA-2D' ||
 * 69.61 || -64.36 || 2.75 || 0 || 0 || 0 || 1.85 || 0.7 || 60.247 || 'AN-698-42027016' ||
 * 69.13 || -55.06 || 6.86 || 0 || 0 || 0 || 5.45 || 4.38 || 247.247 || '3-hydroxy-3-methylglutaryl-coenzymeA-2D' ||
 * 68.93 || -51.76 || 5.78 || 0.95 || 0 || 0 || 4.39 || 4.53 || 213.139 || 'AcetylCoenzymeA-2D' ||
 * 68.66 || -62.47 || 4.54 || 0 || 0 || 0 || 5.66 || 3.9 || 249.725 || '3-hydroxy-3-methylglutaryl-coenzymeA-2D' ||
 * 68.17 || -50.54 || 6.29 || 0 || 0 || 0 || 1.55 || 1.85 || 190.367 || CID15953394-2D' ||
 * 64.42 || -62.58 || 1 || 0 || 0 || 0 || 2.28 || 3.13 || 98.357 || '(5,6-Diphenyl-pyridazin-3-yl)-octyl-amine' ||
 * 64.01 || -54.63 || 4.58 || 0 || 0 || 0 || 3.79 || 3.23 || 202.902 || 'AcetylCoenzymeA-2D' ||
 * 61.3 || -50.07 || 4.87 || 0 || 0 || 0 || 2.92 || 2.42 || 195.229 || 'CoenzymeA-2D' ||
 * 40.92 || -32.38 || 3 || 0 || 0 || 0 || 0.24 || 0.01 || 21.145 || 'aspirin' ||
 * 34.69 || -23.41 || 3.93 || 0 || 0 || 0 || 0.26 || 0.02 || 9.53 || 'alpha-aceto-alpha-hydroxybutyrate' ||
 * 27.58 || -17.98 || 3.2 || 0 || 0 || 0 || 0 || 0 || 16.093 || 'acetoacetic-acid' ||
 * 18.9 || -12.9 || 2 || 0 || 0 || 0 || 0 || 0 || 2.344 || '5-methyl-5H-tetrazole' ||
 * 18.9 || -12.9 || 2 || 0 || 0 || 0 || 0 || 0 || 2.344 || '5-methyl-5H-tetrazole' ||

__**Attempted Docking of NADPH**__ I tried to dock the NADPH so that we could run a virtual on the protein with only the AAC missing. Unfortunately it was too hard to get NADPH to dock without it interfering with the expected position of AAC. We’ll just run this without NADPH and dock with the position provided for AAC. I hope the way that this will work is that since the position was selected for the AAC it will find molecules that bind with the AAC pocket but also hopefully bind partially in the NADPH pocket as to prevent it from properly binding there as well.

__**Coordinates of Substrates to be used by GOLD**__ The coordinates of the substrates used by GOLD to dock the substrates were determined by alignment of 3GK3 Chain B with 3VZS Chain C. A molecule that was deemed a good fit for the main docking point of each substrate was recorded. C/NAP`302/C2D à X = 11.800 Y = 7.656 Z = -6.709 C/CAA`303/C3P à X = 13.213 Y = 15.691 Z = -2.945

__**Determining Best Chain from 3VZS for alignment**__ I want to see which chain from the 3VZS structure would give the substrates in the best position. I will align 3GK3 chain B with all the chains of 3VZS to see which one fits the best. RMS ofChain A .477Chain B .485Chain C .471Chain D .477

So we will use chain C of 3VZS to define the active site of Chain B of 3GK3.

__**Molprobity of 3GK3 Chain B**__ Using the 3GK3 **Chain B** Structure for my protein. After uploading the file

After adding hydrogens, flips, and regenerating H’s

After analyzing the image:


 * **Resolution:** 2.10 Å ||
 * **Clashscore, all atoms:** 8.19 ||
 * **Poor rotamers** 8, 4.55% ||
 * **Ramachandran outliers** 2, 0.84% ||
 * **Ramachandran favored** 226, 94.56% ||
 * **Cβ deviations >0.25Å** 1, 0.46% ||
 * **MolProbity score^** 2.32 ||
 * **Residues with bad bonds:** 0/1815, 0.00% ||
 * **Residues with bad angles:** 0/2453, 0.00% ||
 * **Error in Active Site:** ||

__**Defining Binding Pocket**__ 3GK3 is my protein, but it does not have the substrates bound. 3VZS is a homologous protein that does have the substrates bound. I will first use pocket finder on 3GK3 to see what pockets are predicted. I’ll then superimpose that with 3VZS to see if it finds the correct two pockets. I’ll then superimpose 3GK3 and 3VZS on pymol to define the active site on 3GK3.

**Fig 1.** The pocketfinder results for BpACR. This structure is the BpACR. The grey balloons are the predicted pockets for BpACR. The yellow compounds are the NADPH and the AAC from 3VZS which was superimposed on this molecule and then removed. The NADPH pocket was predicted but the AAC pocket was not predicted by the pocket finder. The other balloon in the back was just an unknown allosteric pocket pictured below.



**Fig 2.** Predicted allosteric binding site by ICM pocket finder.

It turns out that there are some not well defined residues in chain A which happened to fall right around the active site. This may have caused pocket finder to not find the right pocket. This entire thing was attempted once again but this time with chain B. This was the pocket finder result for chain B.

**Fig 3.** Pocket finder prediction of 3GK3. The structure shown is 3GK3 chain B and the yellow compounds are the NADPH and the AAC that came from 3VZS chain B that was superimposed on 3GK3. Chain B was chosen because it did not have the missing residues on the active site that chain A had. This shows better results. You can see the gray bubble includes both NADPH and AAC and the blue bubble is a possible allosteric active site.

So now we can see that using 3GK3 chain B and NADPH and AAC from 3VZS chain B gives us the best result so we’ll use those. Which means we have to do molprobity again….

__**Molprobity of 3GK3 Chain A**__ Using the 3GK3 **Chain A** Structure for my protein. After uploading the file After adding hydrogens, flips, and regenerating H’s





After analyzing the image:


 * **Clashscore, all atoms:** 7.03 ||
 * **Poor rotamers** 6, 3.59% ||
 * **Ramachandran outliers** 0, 0.00% ||
 * **Ramachandran favored** 223, 94.89% ||
 * **Cβ deviations >0.25Å** 0, 0.00% ||
 * **MolProbity score^** 2.16 ||
 * **Residues with bad bonds:** 0/1762, 0.00% ||
 * **Residues with bad angles:** 0/2384, 0.00% ||
 * **Error in Active Site:** ||

__**Molprobity of 3GK3**__ Using the 3GK3 Structure for my protein I did a molprobity analysis of the structure. After uploading the file After adding hydrogens, flips, and regenerating H’s



After analyzing the image:


 * **Clashscore, all atoms:** 9.5 ||
 * **Poor rotamers** 28, 4.08% ||
 * **Ramachandran outliers** 2, .21% ||
 * **Ramachandran favored** 898, 95.03% ||
 * **Cβ deviations >0.25Å** 1, .12% ||
 * **MolProbity score^** 2.31 ||
 * **Residues with bad bonds:** 0 ||
 * **Residues with bad angles:** 2/9651, .02% ||
 * **Error in Active Site:** ||


 * BACKUP!!!! Apparently I need to be doing all of this with only one chain.**

Week 7-8
__**Making Assays More Efficient**__ Now that my enzyme worked I tried to reduce the cost of the assays by making them use less substrate Finally got a "recipe" I was happy with. This decreased the AAC used by half, which is great. Found out I need to be adding 25uL of NADPH to get it up there. You can see the enzyme working in the slope and you can see it run out of substrate when the line goes flat. This still gets about 4 min of enzyme activity which is sufficient.
 * Assay 12:**
 * Figure 4.** Enzyme activation assay of BpACR

This time I tried to increase the amount of AAC and decrease the amount of BpACR by diluting the BpACR with a 1:1 Dilution and then only adding 1uL. 1st peak - Adding NADPH 2nd peak - BpACR You can see some activity but it is very slight, hard to measure. 3rd peak - More BpACR Now you see a better slope but still small. Dip - Added twice as much BpACR This is more like what we were looking for. Will try to get this from the beginning.
 * Assay 11:**
 * Figure 3.** Enzyme activation assay of BpACR

Here I tried to simply reduce AAC, the most expensive substrate, to 50uL as opposed to 200uL. This did not really work. You can see the enzyme work a little bit right after enzyme was added in the prominent peak, but it quickly ran out of substrate AAC.
 * Assay 10:**
 * Figure 2.** Enzyme activation assay of BpACR

First I tried to reproduce the last assay exactly the same way This worked just fine. The results were reproducible.
 * Assay 9:**
 * Figure 1.** Enzyme activation assay of BpACR

Week 5-6
This week I tried to get the enzyme assays to work for the activity of BpACR. Started taking good notes on what was going on during the assay starting on Assay 5. That was better because now I know what every peak and dip was.

__**Assays**__ It finally worked!!! I drastically decreased the amount of enzyme, down to 1uL. This is good because I have plently of enzyme. 1st Read - Buffers 1st Peak - NADPH 2nd Peak - Add a little more NADPH 3rd Peak - BpACR
 * Assay 8:**
 * Figure 5.** Enzyme activation assay of BpACR

This time I thought maybe I was using way way too little AAC, after all I had much more NADPH than AAC. 1st Read - Buffers 1st Peak - NADPH In between (no peak present) - AAC 2nd Peak - BpACR, 20uL It immediately went down, but not in the typical "step", this time it had a curve. I figured it finally worked! Maybe a bit too much enzyme was causing the really steep and unmeasurable curve.
 * Assay 7:**
 * Figure 4.** Enzyme activation assay of BpACR

This time I tried to use a different stored BpACR to see if that worked. When I kept adding stuff and I kept seeing these flat lines, that's when I realized all of these steps were just dilutions. 1st Signal - Buffers 1st Peak - NADPH 2nd Peak - AAC 3rd Peak - BpACR, 30uL 4th Peak - BpACR, 30uL 5th Peak - AAC 40uL Nothing was happening and I kept adding tons of everything. I was thinking this wouldn't work...
 * Assay 6:**
 * Figure 3.** Enzyme activation assay of BpACR

On this assay I finally tried to add the enzyme last. 1st signal - Buffers 1st peak - NADPH 2nd peak - AAC 40uL --> Weirdly starts to go down 3rd peak - Add 25uL of BpACR Last peak - Tried to add more BpACR This assay was flat as Kansas, no activity.
 * Assay 5:**
 * Figure 2.** Enzyme activation assay of BpACR

On this assay I tried to reduce the amount of BpACR being used. I was looking at the steps and thinking that the enzyme might just have been working to quickly to monitor. I still saw the steps since I was using about 25uL of enzyme. I was still adding NADPH and AAC after adding the enzyme which was a problem.
 * Assay 4:**
 * Figure 1.** Enzyme activation assay of BpACR

Week 3-4
This week I spent lots of time trying to design an enzyme assay to test for the activity of BpACR. __**Determination of Minimum Volume for Absorbance Reading**__ In order to use less reagents I checked to see what the minimum amount of liquid was needed to get an accurate absorbance measurement. I did this by taking the absorbance of bromophenol blue, looking for its optimal absorbance wavelength, and then taking absorbance measurements vs. time of that wavelength as I added bromophenol blue to an empty cuvette in 50uL increments. I found that you can get an accurate reading at 250uL, however this is very close to the spectrophotometer's limit and if you dip any lower than this you'll get a very chaotic absorbance pattern. It is recommended to take measurements of cuvettes with atleast 300uL of liquid. I use 400uL of liquid in my assays because using less was not letting me see the absorbance of the materials before I added the enzyme, due to the low liquid level.

__**Enzyme Assay** **Design**__ This enzyme assay was based off of a designed essay found for another Acetoacetyl-CoA Reductase found in a different organism. There are no reliable Km values for either substrate and there is no information at all on the kinetics of my particular target. The following concentrations were proposed for the assay: 60mM Potassium Phosphate Buffer, pH of 5.5 12mM Magnesium Chloride .5mM DTT NADPH - Need to add till absorbance gets to 1-1.5 @340nm .005mM Acetoacetyl-CoA (AAC) 100ng of BpACR Water to 400uL

The amounts of BpACR, AAC, and NADPH were completely experimental. Most of the time spent in these first assays is getting that figured out.

__**Drug** **Dilution**__ The NADPH and the AAC had to be diluted. This definitely required lots of work. Final result: 120 aliquots, 50uL each, of 5mM NADPH in 10mM NaOH stored in -80 117 aliquots, 200uL each, of 0.5mM AAC in 10mM Tris stored in -80

__**Enzyme Assays**__ This assay consisted mostly of messing around with the concentration of NADPH to add. We found that ~12uL of NADPH was doing the job. The enzyme was not working, the curve is the destruction of NADPH due to the pH.
 * Assay 1:**
 * Figure 1.** Enzyme activation assay of BpACR

In this assay I began to add more AAC to see if that would prompt any activity. There was not much change.
 * Assay 2:**
 * Figure 2.** Enzyme activation assay of BpACR

In this assay again we saw no activity when AAC was added. One of the problems with these first assays is that I was adding AAC last and I was adding a ton. This made the characteristic steep drops in absorbance that were caused essentially by an instantaneous dilution. These assays were still inconclusive.
 * Assay 3:**
 * Figure 3.** Enzyme activation assay of BpACR

Week 1-2
After FPLC tubes 32-39 were obtained. Each tube contained 1.3mL of BpACR eluted in sample which means a total of 10.4 mL of sample were obtained. This sample was nanodropped to find the concentration.
 * __Reconcentration and Storage of FPLC__**
 * Figure 1. ** Nanodrop of BpACR in FPLC buffer after reconcentration.

The final volume after reconcentration was 6mL. BpACR has a molar absorptivity of 15595 1/Molar 1/cm which makes this sample's concentration 44.24uM which falls short of the goal of 50-100 uM. These are the results from the google docs spreadsheets:

3mL of this sample was stored in 20% glycerol and placed in the "VDS Proteins" box with orange tape. The rest of the sample was snap frozen and stored in the -80 freezer.

Elutions 1 and 2 were run though the G75 Superdex column to further purify BpACR. The following was the result from the FPLC machine.
 * __FPLC__**
 * Figure 1.** Graph of FPLC of BpACR results. Tubes 32-39 were obtained for reconcentration.

A PAGE gel was made using samples obtained through purification. 1 - ColorPlus Protein Ladder 2 - Cell Lysate before induction 3 - Cell Lysate after induction 4 - Flow Through (Soluble fraction sample was lost) 5 - Wash 6 - Elution 1 (2.35 mg/mL) 7 - Elution 2 (0.53 mg/mL) There was some contamination on the Elution 1 and Elution 2 yields so we proceeded to do FPLC on these elutions.
 * __Characterization Using PAGE__**
 * Figure 1. ** PAGE gel of BpACR using the following samples.

__**Purification of BpACR using Nickel Chromatography**__ After the BL21(DE3) cells were centrifuged, sonicated, and had the soluble fraction extracted from them they were run through a nickel column to purify the BpACR. From this we obtained 2mL of Elution 1 with concentration of 2.35mg/mL and 4mL of Elution 2 with a concentration of 0.53mg/mL.


 * Figures 1&2.** Nanodrop concentrations of Elution 1, trial 1 and trial 2


 * Figures 3&4.** Nanodrop concentrations of Elution 2, trial 1 and trial 2

__**Expression of BpACR in BL21(DE3) cells**__ To express the proteins the cells were grown overnight in the incubator. They were then transferred to 500mL of LB and their absorbance was monitored every 30 minutes to ensure that they have grown enough before we induced expression. Once the absorbance reached .5 at 600nm IPTG was added to induce expression of BpACR.
 * F **** igure 1. ** Log of times at which absorbances were recorded and when IPTG was added.

Week 8
After FPLC tubes 32-39 were obtained. Each tube contained 1.3mL of BpACR eluted in sample which means a total of 10.4 mL of sample were obtained. This sample was nanodropped to find the concentration.
 * __Reconcentration and Storage of FPLC__**
 * Figure 1.** Nanodrop of BpACR in FPLC buffer after collection of tubes 32-39.

The protein was then reconcentrated to reach a concentration between 50 and 100 uM for storage. This was done using a concentrator centrifuge tube spun at 8,000 rpm in increments of 20 min for a total of about 2.5 hours.
 * Figure 2.** Nanodrop of BpACR in FPLC buffer after reconcentration.

The final volume after reconcentration was 2mL. BpACR has a molar absorptivity of 15595 1/Molar 1/cm which makes this sample's concentration 64.76uM which falls between the goal of 50-100 uM. These are the results from the google docs spreadsheets:

1mL of this sample was stored in 20% glycerol and placed in the "VDS Proteins" box with orange tape. The rest of the sample was snap freezed and stored in the -80 freezer.

Elutions 1 and 2 were run though the G75 Superdex column to further purify BpACR. The following was the result from the FPLC machine.
 * __FPLC__**


 * Figure 1.** Graph of FPLC of BpACR results. Tubes 32-39 were obtained for reconcentration.

A PAGE gel was made using samples obtained through purification. 1 - ColorPlus Protein Ladder 2 - Cell Lysate before induction 3 - Cell Lysate after induction 4 - Soluble Fraction 5 - Flow Through 6 - Wash 7 - Elution 1 (2.00 mg/mL) 8 - Elution 2 (0.38 mg/mL) There was some contamination on the Elution 1 and Elution 2 yields so we proceeded to do FPLC on these elutions.
 * __Characterization Using PAGE__**
 * Figure 1.** PAGE gel of BpACR using the following samples.

__**Purification of BpACR using Nickel Chromatography**__ After the BL21(DE3) cells were centrifuged, sonicated, and had the soluble fraction extracted from them they were run through a nickel column to purify the BpACR. From this we obtained 2mL of Elution 1 with concentration of 2.00mg/mL and 4mL of Elution 2 with a concentration of 0.38mg/mL.
 * Figures 1&2.** Nanodrop concentrations of Elution 1, trial 1 and trial 2


 * Figures 3&4.** Nanodrop concentrations of Elution 2, trial 1 and trial 2

__**Expression of BpACR in BL21(DE3) cells**__ To express the proteins the cells were grown overnight in the incubator. They were then transferred to 500mL of LB and their absorbance was monitored every 30 minutes to ensure that they have grown enough before we induced expression. Once the absorbance reached .5 at 600nm IPTG was added to induce expression of BpACR.
 * Figure 1.** Log of times at which absorbances were recorded and when IPTG was added.

__ **Transformation of BL21(DE3) Cells** __ BL21(DE3) cells were transformed with the pNIC-Bsa4+BpACR plasmid. 50ng of plasmid were used for this transformation and one plate was plated with 10uL of cells while another plate was plated with 50uL of cells.
 * Figure 1.** Plates of BL21(DE3) cells transformed with pNIC-Bsa4+BpACR. The plate on the left was plated using 10uL of cells while the plate on the right was plated using 50uL of cells, which is why there are more colonies on the right plate.

Week 7
__**Midiprep of Dh5a containing positive clone**__ Cells from colony 7 were grown and then midi prepped to harvest our pNIC-Bsa4+BpACR plasmid. The final yield was 500uL of 145.3 ng/uL.

Another method of determining if we have a positive clone is by doing a restriction enzyme digest. A restriction enzyme digest was done using HindIII since this enzyme would cut a pNIC-Bsa4 plasmid without an insert in 1 place while cutting a pNIC-Bsa4 plasmid with the BpACR insert in 2 places.
 * __RE Digest of Miniprep Samples__**
 * Figure 1.** Virtual restriction enzyme digest of pNIC-Bsa4 without and insert using HindIII. This cuts the plasmid in one location leading to a gel where a band appears around the 7kb marker on a 1kb ladder.


 * Figure 2.** Virtual restriction enzyme digest of pNIC-Bsa4 with the BpACR insert using HindIII. This cuts the plasmid in two locations leading to a gel where a band appears between the 5kb and the 6kb markers on a 1kb ladder as well as a band above the 500bp marker.

1 - 1kb Ladder 2 - Tube 3 cut pNIC-Bsa4+BpACR 3 - Tube 4 cut pNIC-Bsa4+BpACR 4 - Tube 5 cut pNIC-Bsa4+BpACR 5 - Tube 6 cut pNIC-Bsa4+BpACR 6 - Tube 7 cut pNIC-Bsa4+BpACR 7 - Tube 8 cut pNIC-Bsa4+BpACR 8 - Tube 9 cut pNIC-Bsa4+BpACR 9 - Cut pNIC-Bsa4 without insert 10 - Uncut pNIC-Bsa4 with insert
 * Figure 3.** Restriction enzyme digest of pNIC-Bsa4 with and without BpACR insert using HindIII. The wells contain the following samples:

__**DNA Sequencing of Miniprep Samples**__ Each tube's extracted DNA was sent to DNA sequencing using pLIC forward and reverse primers. This would sequence the region between the pLIC primer locations which is made up mostly of our protein's complete CDS. The following results were returned from sequencing. These forward-reverse pairs correspond only to tubes 3-9 since 1 and 2 kept failing. BpACR's CDS is 747 nucleotides long so we know that something went wrong if we got results less than that number, either the DNA sequencing didn't work well or the insert is incomplete/not present. A blast was done to each one of these results to see which ones had the full CDS of BpACR.

The two tubes that did the best were Tube 5 and Tube 7. Tube 5, however has a deletion on both the forward and reverse sequences. Tube 7, however, was found to contain a 100% positive match of BpACR.

__**Concentration of Miniprep Samples**__ Each of the tubes made from the master plate colonies was miniprepped to extract the plasmid DNA. The following concentrations were found (will just post concentrations since nanodrop images would take up lots of space). Each sample was miniprepped twice since a failed restriction enzyme digest caused me to be short of DNA. Tube 1 - 5.5, 1.8 ng/uL Tube 2 - 5.5, 1.4 ng/uL Tube 3 - 69.1, 91.6 ng/uL Tube 4 - 80.6, 130.0 ng/uL Tube 5 - 33.3, 77.7 ng/uL Tube 6 - 71.7, 75.0 ng/uL Tube 7 - 98.9, 102.6 ng/uL Tube 8 - 53.1, 82.2 ng/uL Tube 9 - 85.9, 88.1 ng/uL

__**Miniprep of Dh5a Containing pNIC-Bsa4+BpACR**__
 * Figure 1.** Master plate containing 9 colonies. Each colony will be tested to see if they contain the correct pNIC-Bsa4 vector + the BpACR insert.


 * Figure 2.** Colony tubes, each growing Dh5a from one of the Master plate colonies.

__**Creating Master Plate**__ A master plate was made containing 9 colonies, each from the following tube cultures: 1 - Tube 2 7/10/14 2 - Tube 2 7/10/14 3 - Tube 1 7/11/14 4 - Tube 1 7/11/14 5 - Tube 2 7/11/14 6 - Tube 2 7/11/14 7 - Tube 2 7/11/14 8 - Tube 2 7/11/14 9 - Tube 2 7/11/14

Week 6
Great work! Good organization, labeling, and analysis. Include pictures of your plates, and move forth and conquer! - Grace 7/15/14

__**Transformation of Dh5a Cells with pNIC-Bsa4+BpACR insert**__ The RE digest of pNIC-Bsa4 with BsaI-HF cut out the SacB gene. The next step was to insert our BpACR gene into this place in the plasmid. This was done by creating sticky ends on the pNIC ends as well as complementary sticky ends on the BpACR insert using T4 DNA Polymerase. Different ratios of pNIC and BpACR were used to attempt to achieve a successful insertion of the gene and consequently a successful transformation of Dh5a cells. When mixed the pNIC and BpACR made a total of 6uL so for example a ratio of 1:2 was made by mixing 2uL pNIC + 4uL BpACR.

On 7/10/14 three plates were made with the following pNIC:BpACR ratios and results: Tube 1 (1:2) - 0 Colonies, taken out of incubator after 2 days Tube 2 (1:1) - 2 Large well defined colonies, taken out of incubator after 1 day Tube 3 (3:4) - This plate is a bit weird, I'd like to get a mentor's opinion on it on Monday. Lots of very very small specks, 1-2 well defined yet small colonies and several very small dots that seem like they could be emerging colonies. Taken out of incubator after 2 days.

Since I had seen little growth the morning of 7/11/14 I went ahead and made 5 more plates with the following pNIC:BpACR ratios and results: Tube 1 (1:2) - 2 Well defined large colonies, taken out of incubator after 1 day. Tube 2 (1:1) - 5 Well defined small colonies, taken out of incubator after 1 day. Tube 3 (3:4) - 1 Fuzzy looking colony, doesn't really look smooth or shiny like all the other colonies I've seen. Taken out of incubator after 1 day. Tube 4 (1:3) - 1 very very small possibly emerging colony. Taken out of incubator after 1 day. Tube 5 (1:6) - 3-4 Very small well defined colonies.

These are all in the 4 degree refrigerator and colonies will be selected for master plate and mini prep on Monday.

__**PCR Cleanup of Digested pNIC-Bsa4**__ The two tubes of digested pNIC were then treated with PCR Cleanup to remove any impurities. The following concentrations were found for each tube.
 * Figure 1.** Nanodrop concentration of pNIC-Bsa4 that has been digested by BsaI-HF and has gone through PCR Cleanup, Tube 1.


 * Figure 2.** Nanodrop concentration of pNIC-Bsa4 that has been digested by BsaI-HF and has gone through PCR Cleanup, Tube 2.

__**RE Digest of pNIC-Bsa4 Using BsaI-HF**__ In order to insert our sequence into the pNIC vector we must cut it first using the BsaI-HF restriction enzyme.
 * Figure 1.** Cutting regions of BsaI restriction enzyme on pNIC-Bsa4. This will cut out the SacB gene, making our cut plasmid capable of surviving on a sucrose coated plate.


 * Figure 2.** NEB Cutter's gel prediction of pNIC-Bsa4 after being digested by BsaI-HF.


 * Figure 3.** Agarose gel run of pNIC-Bsa4 after being digested by BsaI-HF. This run was a success, it came out with the same sizes that were predicted by the NEB cutter and both tubes had their plasmids successfully cut.

1 – 1kb Ladder 2 – RE Digest of pNIC-Bsa4 with BsaI-HF, Tube 1 3 – RE Digest of pNIC-Bsa4 with BsaI-HF, Tube 2

__**PCR Cleanup of BpACR Sequence**__ In PCR Cleanup we purify our double stranded DNA so that we remove any excess primers, nucleotides, DNA polymerase, oils and salts. This was done by centrifuging our PCR squared solution through a special column many times. In the end we eluted the DNA into 50uL of Tris-HCl (Not Elution Solution). A sample was then nanodropped twice and we determined the concentration of the DNA is 129.6 ng/uL.
 * Figure 1.** Nanodrop of BpACR DNA sequence after PCR CleanUp, sample 1


 * Figure 2.** Nanodrop of BpACR DNA sequence after PCR CleanUp, sample 2

__**PCR Squared**__ In PCR Squared the objective was to make many copies of our DNA from secondary PCR so that we could do PCR cleanup and collect it.
 * Figure 1.** Agarose gel of PCR squared for BpACR. The bands are at the correct length because they are all near where 747 nucleotides would be which is the size of the BpACR nucleotide sequence. This means we have successfully amplified the BpACR sequence.

1 – 1kb Ladder 2 – PCR Squared of BpACR Tube 1 3 – PCR Squared of BpACR Tube 2 4 – PCR Squared of BpACR Tube 3 5 – PCR Squared of BpACR Tube 4

__**Secondary PCR**__ In secondary PCR we are joining all of the completed oligos from primary PCR and making a final, complete double stranded BpACR sequence.
 * Figure 1.** Agarose gel of the Secondary PCR of BpACR sequence. The first well is a 1kb dna ladder followed by the secondary PCR sample of BpACR.

1 – 1kb Ladder 2 – Secondary PCR of BpACR

This secondary PCR was successful because what we see is one solid band right between the 1kb and .5kb marker. This is great because the length of the BpACR sequence is 747 nucleotides. This means we now have copies of the completed, double stranded sequence.

__**Restriction Enzyme Digest of pGBR22**__ In restriction enzyme digest we cut up a pGBR22 plasmid with EcoRI and PvuII so that we can determine that we have the correct plasmid.
 * Figure 1.** Agarose gel of pGBR22 cut with EcoRI and PvuII restriction enzymes. The first well contains a 1kb ladder followed by an uncut plasmid followed then by the same plasmid cut by EcoRI, PvuII, and EcoRI and PvuII, respectively.

1 – 1kb Ladder 2 – Uncut pGBR22 plasmid 3 – pGBR22+EcoRI 4 – pGBR22+PvuII 5 – pGBR22+EcoRI+PvuII This restriction enzyme digest was a success because of the different size bands that we are seeing. The pGBR22 plasmid has a length of 3,750 nucleotides. In well two we see that the plasmid goes past the 3,000 nt mark which would mean the plasmid is shorter than the sequence. What is happening is that since the plasmid is uncut it coils up as it passes the agarose and effectively acts as a shorter nucleotide sequence. When cut once by EcoRI as seen in well 3 we see that it is between the 3kb and 4kb mark which is fantastic. We also see in well 4 that it is cut twice and as a result we get 2 fragments, one slightly less than 3kb and one slightly more than 1kb which if we add up we can see would end up somewhere around 3.75kb. We see this again in Well 5 when it is cut three times, all of the fragment sizes seem to add up to 3.75kb which is great because that is the original size of the plasmid. We also notice the smaller bands are also lighter, this is because there is less ethidium bromide intercalated in the nucleotides because there are less nucleotides.

Week 5
__**Storage of FtHap**__ The FtHap that was collected from the FPLC was then stored. This was done by re-concentrating the protein to a concentration of 64.3 mM using a concentrator centrifuge tube. This was much easier said than done, it took much of the day to get the sample down to that concentration but when we finally did it we stored the remaining solution in two different forms. Half of the solution was stored with 20% glycerol into the -20 degree freezer. The other half of the solution was then snap freezed using liquid nitrogen and stored into the -80 degree freezer. These proteins can later be used for assays to determine if they are active or protein binding assays.

__**Primary PCR**__ With our oligonucleotides mixed together the first step is to fill in the gaps between the oligos. This is done in primary PCR. This will result in many DNA fragments that are completed and as a result if we run this through a gel we will expect a smear to appear because of the many different sizes of the completed oligos. This was done and stored, next week we will move on to secondary PCR which will piece together all of the oligos into a completed sequence.
 * Figure 1.** Agarose gel of primary and secondary PCR’s. The first well contains a 1kb dna ladder and the following gel contains Haley’s primary PCR sample. The 4th from last gel contains Luis’ 1st primary PCR followed by his 1st secondary PCR followed by his 2nd primary PCR and finally Charina’s 1st primary PCR. All the PCR’s except for the well 9 primary PCR failed perhaps because we used thermopol buffer on accident instead of Q5 reaction buffer. The streak on well 9 shows that the oligos have been completed and are of many different lengths for Luis’ target, BpACR. The size of BpACR is 747 bp so the streak is darkest in this range.

1 – 1kb Ladder 2 – Haley primary PCR 3 – Empty 4 – Empty 5 – Empty 6 – Empty 7 – Luis 1st Primary PCR (BpACR) 8 – Luis 1st Secondary PCR (BpACR) 9 – Luis 2nd Primary PCR (BpACR) 10 – Charina 1st Primary PCR

__**Oligo Mixing**__ PROJECT BpACR (Burkholderia psuedomallei Acetoacetyl-CoA-Reductase) HAS SET SAIL! Zain and I are pretty pumped that we finally started on our project. To begin, we made an oligo mix. The DNA template for the coding sequence of BpACR was delivered to us in 18 different oligonucleotide fragments. We mixed these fragments together so that each oligonucleotide had a concentration of 1uM.

__**FPLC of FtHap**__ We used the FPLC machine in the biotech lab to purify our FtHap protein. FPLC sorts proteins by size in a column filled with buffer and then dispenses about a mL of weight-sorted solution into many collection test tubes. The proteins are sorted through the G75 using "wuffle balls" which let heavy proteins flow through quickly but the smaller the protein the more it will get caught within the resin and as a result it is dispensed last by the FPLC machine. There are three standards used create 3 peaks, one at kDa, 44kDa, and 25kDa respectively and these dotted peaks are used so that we can estimate the protein size of our purified protein which is represented by the solid line peaks. We can see in this picture there are about 3 solid peaks, these are all proteins but in order to find our FtHap protein we used the standard curve to determine that the highest peak(the 2nd peak) is our FtHap protein and the other peaks are either contamination or another protein. The red lines show in which tubes the purified protein was dispensed into, in this case tubes 31-36. This sample was collected for re-concentration and storage.
 * Figure 1.** FPLC result of the purification of FtHap. The solid blue line represents the UV absorbance of the solution that was being dispensed into the collection tubes. The absorbance is low when there is only buffer being dispensed and it peaks when there are proteins which do absorb these wavelengths especially in the 280nm range. This absorbance is compared to a standard that is represented by the dotted blue line.

__**Designing and Ordering Tail Primers**__ We designed Tail primers for secondary PCR of our oligo mix. We will use these tail primers to amplify our coding DNA sequence for our protein, BpACR. The length of the Forward Primer was modified to reach a melting temperature within .5 degrees for the forward and reverse primer at a 2mM Mg2+ concentration. The melting temperature will be 71.6-72.1 degrees Celsius which is a bit high but it should work. The melting temperature is determined by the GC content of the primer, a higher GC content will result in a higher melting temperature.

__**PCR of pNIC-Bsa4**__ As another exercise in preparation for future PCR's we did PCR on pNIC using pLIC-forward and pLIC-reverse primers. This effectively amplified the SacB region of the pNIC-Bsa4 which is a gene that is used for negative selection on 5% sucrose. The reason for this gene is that it inhibits the growth of any cells that were transformed but did not get a gene inserted by coding for a protein that is sensitive to sucrose.
 * Figure 1.** PCR agarose gel for pNIC-Bsa4. Lane 1 contains the 100 bp DNA ladder and lane 10 contains a 1 kb DNA ladder. The absence of sample in lane 7 is due to a hole in the well through which all of the sample fell through when being loaded. Lanes 2-5 contain PCR DNA samples A-D for Brianna and lanes 6-9 contain PCR DNA samples for Luis. There is evidence of some contamination because of the presence of two bands in almost every lane. The samples are around the right size because the band is slightly below the 1000bp mark which is around the size of the SacB gene.


 * Lane 1:** 100 bp DNA Ladder
 * Lane 2:** 0.0463 ng DNA template (Brianna)
 * Lane 3:** 0.463 ng DNA template (Brianna)
 * Lane 4:** 4.63 ng DNA template (Brianna)
 * Lane 5:** Control, No DNA template (Brianna)
 * Lane 6:** 0.0463 ng DNA template (Luis)
 * Lane 7:** 0.463 ng DNA template (Luis)
 * Lane 8:** 4.63 ng DNA template (Luis)
 * Lane 9:** Control, No DNA template (Luis)
 * Lane 10:** 1kb DNA Ladder

Week 4
__**Making a PAGE gel for**__ __**characterizatio****n of** **FtH****ap samples, round 2**__ Once again we made a PAGE gel from scratch to characterize our FtHap samples. Once completed our gel wells contained the following samples: Well 1: Protein Ladder Well 2: Cell lysate before induction step Well 3: Cell lysate after induction step Well 4: Soluble Fraction Well 5: Flow through Well 6: Wash Well 7: Elution 1 Well 8: Elution 2

The gel only came down about 1/3 of the length of the gel instead of coming down about all of the distance. This may have been because when we made our gel we had to place another layer of the initial bottom portion of the gel and this caused a sort of fault line to form that may have prevented the bands from traveling. This gel shows some a streak of contamination particularly in the elution 1 sample but the elution 2 sample shows more signs of having a single pure band. We will be running the elution 1 from purification together with Charina's elution 1 to conduct FPLC.

__**Making a PAGE gel for characterization of FtHap Samples**__ We made a PAGE gel from scratch to characterize our FtHap samples. Once completed our gel wells contained the following samples: Well 1: Protein Ladder Well 2: Cell lysate before induction step Well 3: Cell lysate after induction step Well 4: Soluble Fraction Well 5: Flow through Well 6: Wash Well 7: Elution 1 Well 8: Elution 2

The expected results if everything went perfectly were: Well 1: A staggered ladder containing reference molecular weights for proteins Well 2: A large streak that is lacking darkness in the area where our harvested protein is to be found. This is because we haven't induced expression at this point so we should not have much of our target protein since we are trying to give our cell a chance to grow into a large culture before we use resources to make protein. Well 3: A large streak that is also dark in the area where our harvested protein is to be found. This should have all of the soluble proteins of the cell. Well 4: A large streak that is missing a considerable amount of area and darkness. This flow through step expels any protein that is not bound to the Ni-NTA beads but is still soluble. There are proteins other than our target that are still bound to the Ni-NTA. Well 5: In this step we add a slight amount of imidazole to remove any protein that is loosely bound to the Ni-NTA. We should see a light band or streak depending on how much protein was loosely bound. Well 6: A dark, single band indicating the size of our FtHap. Well 7: Perhaps a lighter band still right at the same level as our well 6 band.

Our gel did not run very well. It seems that it was only able to get halfway through the gel and then the streaks sort of dissipated. This could be because of the way the gel was made, the first layer only came up about halfway through the gel and the second layer made up the other half instead of having the first layer make up 3/4 of the gel. We will repeat this experiment with more sample.

__**Purification of FtHap protein from Sonicated BL21(DE3) Cells**__ From the sonicated cells FtHap was purified using nickel affinity chromatography. Since the protein is soluble we extracted the supernatant from the lysed cells and ran that through a column containing nickel attached to beads that bind to a his-tag on the protein. This way we collect our protein at the bottom. Nickel chromatography yielded a 15mL conical tube with an FtHap concentration of .92mg/mL.


 * Figure 1.** Column used for protein purification containing Ni-NTA resin for nickel affinity chromatography.


 * Figure 1.** Concentration of FtHap from Sample 5 Elution 1 is .92 mg/mL measured at 280nm


 * Figure 2.** Concentration of FtHap from Sample 6 Elution 2 is .61 mg/mL measured at 280nm

__**DNA Sequencing of pNIC made from Midi prep**__ To verify that I had harvested the original pNIC I intended on making from my Dh5a cells I sent a sample of this collected plasmid to DNA sequencing using pLIC forward and reverse primers that were provided in our lab. The sequencing was a success, the reads were excellent with my pLIC-forward sample reading 1022 nucleotides and my pLIC-reverse sample reading 999 nucleotides.


 * Figure 1.** 1022 out of ~1000 Nucleotides were sequenced for the pNIC plasmid. This was sequenced using pLIC-for primer.


 * Figure 2.** 999 out of ~1000 Nucleotides were sequenced for the pNIC plasmid. This was sequenced using pLIC-rev primer.

After a DNA sequence analysis it was determined that the sequence is indeed the same sequence recorded for pNIC-Bsa4.

__**Midi Prep of pNIC+Bsa4 DH5a Cells**__ I repeated the expression of pNIC+Bsa4 in DH5a cells for the purpose of purifying this plasmid for use in future procedures with my target. With this midi prep I collected 1mL of 77.5 ng/uL pNIC plasmid in TE buffer.
 * Figure 3.** Nanodrop of pNIC-Bsa4 plasmid made in Dh5a cells and purified by midi prep. The concentration was determined to be 75.3 ng/uL


 * Figure 4.** Nanodrop of pNIC-Bsa4 plasmid made in Dh5a cells and purified by midi prep. The concentration was determined to be 77.6 ng/uL

__**PCR of pGBR22**__ The day finally came, the PCR of pGBR22 finally worked on my 3rd attempt. This was accomplished using M13 forward and M13 reverse primers. Once again we prepared 4 tubes for PCR and they consisted of the following: Tube A: .3ng of pGBR22 Tube B: 3ng of pGBR22 Tube C: 30ng of pGBR22 Tube D: Control tube, no DNA in this tube. Troubleshooting: To find out what was going wrong in previous PCR's we analyzed what we could from our gels. On my first PCR I got nothing but a few very faint contamination smears, telling me I had to be more sterile in my PCR technique. In response to this I payed particular care to keep my PCR sterile on the second round and I remade my template dilutions to make sure that they were not part of the problem. Again nothing showed up but I did not get any contamination bars so this time I used a different set of diluted primers. This must have done the trick because on the next PCR attempt I was able to get the bars I was looking for.

__**Analyzed DNA Sequencing Results f****or** **pGBR22**__ We went to the computer lab and learned the procedure for analyzing DNA sequencing results and using them to verify that they plasmid that you have in lab is the same as the plasmid that you originally intended on replicating. This procedure involved using NCBI's blast to compare your sequence to those of other proteins found in the database and creating a pairwise alignment to see if the sequence matches up perfectly to any of the known sequences. We then inserted the sequence for the GBR gene into a pGEMT backbone.

Week 3
__**PCR of pGBR22**__ Another way to make copies of DNA is to do PCR on them. PCR stands for Polymerase Chain Reaction and basically what happens is you make copies of DNA and then use those copies as a template to make more copies. We used M13 forward and reverse primers for this PCR. Preparing the PCR reaction tubes took an hour or two since it was our first time but we've been reassured by our mentors that we will soon be PCR pros that could get a reaction rolling in 15 minutes! We prepared 4 tubes for PCR and they consisted of the following: Tube A: .3ng of pGBR22 Tube B: 3ng of pGBR22 Tube C: 30ng of pGBR22 Tube D: Control tube, no DNA in this tube, After PCR we stored the tubes in the -20 freezer in our lab.
 * Figure 5.** Our first PCR!!

The next day we ran gels of the PCR to determine if our pGBR22 was copied correctly.
 * Figure 4:** Agarose gel for pGBR22 samples from PCR run. 1st lane contains the ladder while the subsequent 4 lanes contain 3 samples and a control channel, subsequently. There are no bands and the streaks on well 4 (sample 3) suggest contamination.

In theory the 2nd, 3rd, and 4th lanes should have 1 solid band, any more and we know that we had contamination, DNA fragments that are not pGBR22 are present. These results show a few bands in lane 4 which suggest there was contamination. There is also a lack of solid bands so we know we didn't successfully replicate pGBR22. We are working on repeating this protocol as soon as week 4 starts.

__**Midi Prep of pNIC+Bsa4 DH5a Cells**__ The next step with our pNIC+Bsa4 DH5a cells was to purify the plasmids that we had been making so many copies of inside of the DH5a cells. We began by centrifuging the DH5a cells in the large centrifuge found in our lab and then removing the supernatant liquid which was mostly just LB. We resuspended the pellet in P1 buffer and then proceeded to do the full Midi prep which consisted of washing the cells with 2 buffers, lysating the cells, filtering out the solid waste, precipitating the DNA, and finally collecting the precipitated DNA with TE buffer. I learned the following from this procedure: I learned the last one the very hard way. I'm embarrassed by this but I accidentally deposited all of my purified plasmids into my waste container because I didn't realize the TE buffer collected the DNA. Because of this I'll have to repeat midi prep next week so that I can have this plasmid backbone ready to go for my protein target.
 * Keep a timer nearby to be able to keep track of the waiting times easily and so that you don't get your waiting times mixed up with any of your partners
 * Read all the labels of what you are putting into your midi prep very very carefully because they all look almost identical and you can really mess up your midi prep this way
 * Never pull back on the plunger of the syringe until you have removed the QIAprecipitator from the syringe
 * Collection tube =/= waste tube.

In order to purify protein we had to grow our overnight culture a little more so that we would have a more significant yield. This particular cell does not express the protein we transformed into it unless we add IPTG so first we want to grow a substantial amount of cells before we begin using the cell's resources to make FtHap. We did this by taking 10mL of our sample and adding it to 500mL of LB in a 2L flask, then slowly adding more while monitoring the absorbance of the container with a Vernier Spectrophotometer ("Chipper"). Once we reached an absorbance measure at 600nm of .1 we placed it in the incubator to grow. We continued to monitor the absorbance as this culture grew and we planned on adding IPTG once the absorbance reached .5, however, after 3 hours we added IPTG when our culture had an absorbance of .367 because we were running short on time. This means we won't have as a significant yield of protein, but this was done since this is practice and we needed to move on to other activities.
 * __Protein Expression of FtHap__**

We spun down the culture of cells we had grown in the very large centrifuge found in the DIY lab. This produced a pellet that weighed 9.33 grams. This pellet contains (still intact) cells while the supernatant liquid is mostly LB media. The purpose of this was to separate the cells from the LB media and store them for sonication the next day. We resuspended the cells in 10mL of Lysis/Sonication Buffer.

The next day we took the cells we had resuspended in buffer and sonicated them in the sonicator found in the BioTech lab. This process is super cool and consists of lysing cells using vibrations that we turn on periodically using the sonicator. The end result of this is a tube filled with lysed cells. The next step will be to purify the protein that is now found dissolved in the supernatant liquid.

I learned several important skills in this process including:
 * How to grow a large culture that we can monitor absorbance for over a period of time
 * How to use the formula found in the excel spreadsheet to predict the amount of time it takes for a culture to reach an absorbance value of .5
 * How to induce the expression of proteins in BL21 (D3) cells
 * Introduction to the giant centrifuge and how to properly use it.
 * Introduction to the sonicator found in the BioTech lab and how to properly use this delicate tool without breaking it.

__**Overnight Culture of FtHap+pNIC+Bsa4 BL21 (D3) Cells**__ After my 3rd overnight culture of the week I think I can do this in my sleep. Along with Andrei I was assigned FtHap BL21 (D3) cells to culture overnight. The purpose of this culture is so that we could purify this protein. We grew two colonies in two 125mL Erlenmeyer flasks each with 50mL of LB. We added kanamycin to make sure that only transformed cells could grow in this culture. One thing to note about this culture is that we were very tight on room in the incubator so we were told it was OK to grow these cells in 125mL flasks even though standard procedure says we should grow cells in containers atleast 4 times their size for proper aeration. This turned out to be fine as the cells grew just fine.
 * Figure 3.** Overnight culture of FtHap+pNIC+Bsa4 BL21 (D3) cells to be used for protein expression and purification. They were nice and cloudy in the morning so we moved on to protein expression.

__**Overnight Culture of pNIC+Bsa4 DH5a Cells**__ In order to have some cell culture to do Midi prep on I grew the pNIC+Bsa4 DH5a cells overnight. This included growing taking a colony from these cells and growing them in 160mL of LB media in an Erlenmeyer flask overnight. We added Kanamycin to make sure that only transformed cells could grow in this culture. This worked just fine and in the morning I had a cloudy Erlenmeyer flask that I could use the cells from for Midi prep.

__**Overnight Culture of YopH+pNIC BL21 (D3) Cells**__ We were all instructed to make an overnight culture growth of the cells we had transformed last week. This included taking a colony from our plates and having it grow in 160mL of LB media in an Erlenmeyer flask. We added kanamycin to make sure that only transformed cells could grow in this culture. The purpose of this was so that we could have some cells to do Midi prep to so we could collect plasmids. However, I was one of the few people that was assigned to transform BL21 (D3) cells instead of DH5a cells so this culture turned out to not be necessary.

__**Making Sucrose+LB+Kanamycin Agar Plates**__ In preparation for future VDS activities we made 23 Suc+LB+Kan Agar plates. Why 23? We made 25 and 2 plates fell victim to bubbles. Even though making these plates is a straightforward and standard protocol I did get to refresh some important techniques including:
 * How to interpret Weight/Vol percent solutions
 * How to properly use a bottle top filter
 * Anything liquid being autoclaved should be in a container at least 2 times its volume in size
 * When adding Kanamycin our lab uses a 50 ng/uL kan dilution which means we can always add this solution in a 1uL Kan : 1mL of liquid we want to apply this antibiotic to
 * How to pour agar plates without creating bubbles

__**Sequencing of pGFP redo**__ Last week we attempted to determine the sequence of pGFP using M13 for and rev primers and we were not successful. This week we once again tried to determine the sequence of pGFP using SP6 and T7 primers instead and once again it didn't work. There were a few things that are of concern with this sequence run, however: Charina also is in the same situation as Andrei and me. Her first attempt failed and she used this pGFP from 2010 to try to redo the sequencing and she might be getting her results this coming week.
 * We couldn't find the same pGFP we used earlier to save our lives, so we used another unverified pGFP that was made in 2010, so it is possible this sample does not even have pGFP for us to sequence since it is unverified.
 * This pGFP was not nanodropped, we went off the 79.4 ng/uL reading on the tube.
 * This time we used in-house primers (option A) while the last time we used our own primers. It is possible that we did not do a good job of adding primers the first time and that M13 for/rev are in fact the right primers to use.


 * Figure 1.** 92 out of ~1000 Nucleotides were sequenced for the pGFP plasmid. This was sequenced using T7 primers.


 * Figure 2. 339** out of ~1000 Nucleotides were sequenced for the pGFP plasmid. This was sequenced using SP6 primers.

Week 1 and 2
Transformation efficiency = 14 transformants/ug of plasmid.
 * Figure 1.** Plate A containing 1 ng of YopH+pNIC in competent BL21-D3 cells grown in a kanamycin and SOC coated agar plate. A total of 14 colonies grew on this plate.


 * Figure 2.** Plate B containing 5 ng of YopH+pNIC in competent BL21-D3 cells grown in a kanamycin and SOC coated agar plate. No colonies grew on this plate for some reason.


 * Figure 3.** Plate C containing 25 ng of YopH+pNIC in competent BL21-D3 cells grown in a kanamycin and SOC coated agar plate. A total of alot ~392 colonies grew on this plate due to the higher plasmid concentration and as a result higher transformation efficiency. Transformation efficiency = 15.68 transformants/ug of plasmid.


 * Figure 4.** Plate D containing 0 ng of YopH+pNIC in competent BL21-D3 cells grown in a kanamycin and SOC coated agar plate. No colonies grew on this plate as expected.


 * Figure 5.** Sequencing FAIL. 49 out of ~1000 Nucleotides were sequenced for the pGFP plasmid. This was sequenced using M13-for and M13-rev primers. Andrei and I are trying again using T7 promoter and SP6 promoter. More on that next week!