Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Abstract

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

Introduction

Bacteria sense population density and the type of cells nearby using a cell-cell communication process called quorum sensing (QS)¹. Diverse clades of bacteria use QS to control various behaviors, such as motility, biofilm formation, virulence factor secretion, and more. The proteins and signals involved in QS differ widely among bacteria. In Vibrio species, the QS signaling system predominantly uses membrane-bound hybrid histidine-kinase receptors that recognize specific cognate small molecule signals called autoinducers² (Figure 1). These receptors control the flow of phosphate through the system to a response regulator that transcribes small RNAs. The production of sRNAs alters the production of the master quorum sensing regulator, which is defined as a conserved group of proteins collectively called LuxR/HapR³. Thus, at low cell densities, the mRNA encoding LuxR/HapR is degraded via sRNA targeting, and at high cell density, LuxR/HapR protein is produced at maximal levels (reviewed in Ball et al.³).

The LuxR/HapR group of proteins belongs to the large group of TetR proteins, which is defined by the presence of a helix-turn-helix in the DNA binding domain, formation of a functional homodimer, and typically the inclusion of a ligand binding domain⁴. The Vibrio LuxR/HapR proteins fit all these criteria, though a ligand has not yet been identified. The LuxR/HapR protein in all studied Vibrio species controls numerous downstream behaviors, many of which are known to be important in pathogenesis: production of biofilms, proteases, cytotoxins, hemolysins, type III secretion, and type VI secretion complexes, and more³. Deletion of the LuxR/HapR protein leads to a decrease or loss of virulence in host systems^5,6,7, leading to the hypothesis that inhibition of these proteins is a viable strategy for counteracting disease progression. Vibrio species cause vibriosis disease in marine organisms, including fish, shellfish, and corals, as well as in humans who contact or ingest certain species.

Previous research has identified a panel of thiophenesulfonamide compounds that specifically bind to the ligand binding domain of LuxR/HapR proteins in multiple Vibrio species to block their function^5,8,9 (Figure 1). Using a reporter screen in E. coli, the compounds were identified and subsequently tested in the native Vibrio, which showed a high correlation between the effect in the heterologous E. coli and the efficacy in the Vibrio⁹. These compounds are simple to synthesize in a single step, making them ideal for small library synthesis in the context of a chemical biology lab course. A three-week course-based undergraduate research experience (CURE) that was designed around this molecular scaffold has been previously reported⁸. This three-week module has been further optimized, streamlined, and broadened in this year-long CURE designed to target the inhibition of LuxR/HapR proteins in diverse Vibrio species.

Protocol

The details of the reagents and the equipment used for the study are listed in the Table of Materials.

1. Design and synthesis of thiophenesulfonamide libraries

NOTE: Thiophenesulfonamide inhibitors such as 3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole (PTSP) are synthesized via the one-step base-promoted condensation^8,9, as shown in Figure 2. To design new compound libraries for study, researchers must procure appropriate amines and sulfonyl chlorides and follow the steps summarized below. If the structure of the amine differs greatly from the aromatic pyrazole derivative, then alternative reaction conditions may need to be identified by searching the literature. This procedure is robust, and bases such as sodium hydroxide, triethyl amine, and pyridine have also worked well. If this is performed in a course, students may be given the freedom to choose their own procedure with likely favorable outcomes.

1. Synthesis and isolation of PTSP
   NOTE: This can be completed in one 3 h lab period.
   1. Dissolve 822 mg of phenylpyrazole (5.7 mmol of the amine, 1.5 eq.) in 15 mL of tetrahydrofuran in a 50 mL round bottom flask with a stir bar.
   2. Slowly add 306 mg of sodium hydride (60% in oil, 7.65 mmol, 2 eq.) to create a suspension. Let this suspension stir on a stir plate for 10 min.
   3. In a vial, prepare a solution of thiophenesulfonyl chloride (3.82 mmol of the sulfonyl chloride, 1 eq.) in 5 mL of THF.
   4. Add the solution of sulfonyl chloride prepared in step 1.1.3 to the suspension prepared in step 1.1.2 and allow the resulting suspension to stir for an additional 30 min.
   5. Add 20 mL of DI water to the reaction mixture in the 50 mL round bottom flask.
   6. Transfer all contents of the reaction flask except the stir bar into a 250 mL separatory funnel¹⁰.
   7. Add 20 mL of ethyl acetate (EtOAc) to the separatory funnel and shake.
   8. Remove the bottom (aqueous) layer and set aside.
   9. Remove the top (organic) layer and set aside in a 200 mL Erlenmeyer flask.
   10. Return the aqueous layer to the separatory funnel and extract with 20 mL of ethyl acetate.
   11. Repeat steps 1.1.8 and 1.1.9.
   12. Return the combined organic layers to the separatory funnel and wash with 20 mL of saturated NaCl (brine).
   13. Remove the bottom (aqueous) layer and set aside.
   14. Drain the top (organic) layer back into the Erlenmeyer flask.
   15. Dry the combined organic layers over MgSO₄ in the Erlenmeyer flask and filter into a 250 mL round bottom flask using qualitative filter paper¹⁰.
   16. Remove the organic solvent on a rotary evaporator.
2. Analyze the crude reaction mixture by ¹H NMR to ensure the product has formed¹⁰.
   NOTE: The time necessary for this step will depend on the student's comfort level with NMR spectroscopy.
3. Purify the product by silica gel column chromatography (10:1 hexanes: ethyl acetate)¹⁰.
   NOTE: This can be completed in one 3 h lab period.
4. Characterize the product spectroscopically.
   NOTE: The time necessary for this step will depend on the student's comfort level with NMR spectroscopy.
   NOTE: Characterization data for 3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole (PTSP)¹⁰:
   ¹H NMR (400 MHz, CDCl ₃ ): δ 8.11 (d, J = 2.8 Hz, 1H), 7.88 - 7.79 (m, 3H), 7.68 (dd, J = 5.0, 1.4 Hz, 1H), 7.44 - 7.31 (m, 3H), 7.07 (dd, J = 5.0, 3.9 Hz, 1H), 6.71 (d, J = 2.8 Hz, 1H).
   ¹³C NMR (101 MHz, CDCl ₃ ): δ 157.17, 136.84, 135.40, 135.36, 132.55, 131.28, 129.36, 128.72, 127.79, 126.47, 106.90.
   HRMS (ESI): Calculated for C₁₃H₁₀O₂N₂NaS₂ [M+Na⁺]: 313.0076. Found: 313.0078.

2. Structural modeling to predict the binding of thiophenesulfonamides to Vibrio LuxR/HapR regulator

NOTE: This protocol utilizes the web-based version of AutoDock Vina called Webina¹¹ to dock small molecule inhibitors (PTSP in this case) into the ligand binding pocket of the LuxR/HapR homolog called SmcR from Vibrio vulnificus¹². The outcomes of this protocol are (1) calculated binding affinities and (2) a structure file that can be opened in PyMol or a related program to visualize the ligand-protein interactions. This protocol can be adapted for any small molecule and any protein with a ligand binding pocket. It takes minutes to dock into SmcR because the search area for this receptor is defined below. If the search area needs to be defined for a new receptor (steps 2.15-2.20), this can be completed in one 3 h lab period. Once the search area is defined, subsequent dockings will take minutes.

1. Download the AutoDock Tools. Select the appropriate file for the operating system and install it on the computer.
   NOTE: This protocol utilizes a web-based version of AutoDock Vina. The docking can also be performed locally if this program is installed (see the manufacturer's instructions).
2. Download the structure for apo SmcR.
3. Navigate to the Protein Data Bank.
4. In the search bar, enter the PDB ID 3KZ9.
5. Click on the Download Files from the dropdown menu toward the top right corner of the page and select pdb format.
6. After downloading the file, save it to a new folder dedicated to the docking work.
7. Create a .mol structure file of the small molecule inhibitor (PTSP).
8. Navigate to molview and click on the trash can icon to remove the structure that is there.
9. Draw PTSP in the structure window.
10. Click on 2D to 3D.
11. Click on Tools → export → MOL file.
12. A file titled "Molview.mol" will be downloaded to the computer. Give the file a name that makes sense and save it to the folder that contains the protein pdb file.
13. Define the search area.
14. Define the area in Webina as the binding pocket of the protein (SmcR) to facilitate docking in the correct region.
    NOTE: Coordinates for SmcR are provided and generated using the following protocol. If SmcR is being used, then steps 2.15-2.20 can be skipped. The protocol can be adapted for any protein with a known ligand binding pocket. Coordinates: center_x = 17, center_y = 40, center_z = 56, size_x = 12, size_y = 12, size_z = 12.
15. In AutoDock Tools, click on File → Read Molecule and open the pdb protein file downloaded in step 2.5.
16. Navigate to the dashboard and click on the blue arrow next to the name of the protein. This will bring up a list of chains. Click on the blue arrow next to one of the protein chains.
17. To highlight the amino acids of the binding pocket, find the amino acid of interest in the list that appeared. Next to the amino acid, go to the triangle in the Cl (color) column at the far right. Click on the triangle and select by rainbow.
    NOTE: Use this method to highlight the following amino acids:¹² Phe75, Phe78, Leu79, Ile96, Met100, Trp114, Phe129, Asn133, Gln137, Val140, Ala163, Phe166, His167, Cys170 (If Met100 is missing, this can be skipped).
18. Next, click on Grid → GridBox. A box should appear over the protein structure with a red, green, and blue face. Before making any changes, change Spacing (angstrom) to 1. This will ensure the box is correctly scaled for the next steps.
    1. Then, change the Center Grid Box <offset> dials to translate the box in the x, y, and z directions to place it over the highlighted amino acids. Click and drag on the protein to rotate the structure in three dimensions.
       NOTE: This will help ensure that the box is in the correct position. If necessary, use the top three dials for the number of points to change the size of the box.
19. Once the box position and dimensions are set, click on File → Close Saving Current.
20. In the menu, click on Grid → Output → Save GPF. Name the file and save it in the same folder as the two structure files. This GPF file has the box coordinates.
21. Perform "Docking" in Webina to generate a calculated binding energy.
22. Open the folder that contains the .mol ligand file and the .pdb protein file. Webina requires .pdbqt files, and will do the file conversion.
23. Navigate to Webina.
24. Receptor: Drag over the file downloaded from the pdb called 3kz9.pdb. Click on Add hydrogen atoms, and click on convert. Click on Ok on the next pop-up to work with a monomer.
25. Ligand: Drag over the .mol file for PTSP (or other small molecule inhibitor). Ensure no boxes are selected and click on Convert. Leave "Correct Pose" empty and scroll down to the Docking Box. Enter the appropriate box parameters (either those listed above for SmcR or a set that was generated by the protocol in steps 2.15-2.20 for a different protein).
26. Click on Start Webina and wait.
27. A series of calculated binding energies will be reported [Affinity (kcal/mol)] directly below a visualization of the docked molecule. Ensure that the first value is the most negative.
28. To visualize the docked ligand in pymol or another similar program, download the Output PDBQT File by clicking on the appropriate download button. This file is just the ligand (PTSP) in the docked coordinates.
29. Visualize the docked ligand-SmcR complex in PyMol¹³.
    1. Download and open PyMol.
       NOTE: Full-time students and educators may receive a PyMol License free of charge. When asked for a license after starting PyMol for the first time, click on Buy License. Select Student/Teacher.
    2. Open the pdb file called 3kz9.pdb.
    3. Open the pdbqt output file created by Webina.
    4. Visualize the best conformation (the one with the lowest binding affinity) by clicking on the small pointing looking arrows at the bottom right of the PyMol screen. The first conformation will be the most favorable and will be showing when the file is opened.
    5. Manipulate the protein with the docked ligand in PyMol using standard commands¹⁴.
       NOTE: The previous study by Newman et al. compared the predicted binding affinities of several compounds determined by Webina to in vitro and in vivo assays⁹. These binding affinities serve as references for compounds that are likely to be good inhibitors with high affinities (which correlate to lower kcal/mol numbers) for the binding pocket. An example of the Webina output data is included in Figure 3A,B for PTSP binding in the pocket of SmcR.

3. Biological assessment of thiophenesulfonamides in quorum sensing inhibition

NOTE: The procedure specifically for assaying the Vibrio campbellii protein LuxR is described here. However, this procedure can be adapted for use with any of the Vibrio LuxR/HapR proteins, all of which are available on ectopically replicating plasmids that are compatible with the reporter plasmid pJV064⁹. The "red-green screen" assay is performed in a heterologous bacterial background using E. coli cells containing the two plasmids. The LuxR/HapR expression plasmid (conferring kanamycin resistance) is available expressing different LuxR genes (Figure 4A). The pJV064 plasmid (conferring chloramphenicol resistance) encodes a gfp gene under the control of a LuxR-activated promoter and a mCherry gene under the control of a LuxR-repressed promoter (Figure 4A). Both promoters were chosen due to their identification as LuxR-regulated genes with large changes in expression in vivo¹⁵. The LuxR/HapR E. coli strains and plasmid pJV064 have been published previously^9,15.

1. Liquid media preparation: Prepare 1 L of LB medium.
   1. Weigh out 10 g of NaCl, 10 g of bactotryptone, and 5 g of yeast extract.
   2. Mix the components in a beaker or graduated cylinder using a stir plate.
   3. Bring the total volume to 1 L using graduated cylinders.
   4. If desired, aliquot into 100 mL per bottle before autoclaving.
   5. Autoclave for 15 min for each 1 L of media. Alternatively, if an autoclave is not available, an Instant Pot may be used¹⁶.
2. Strain inoculation (Day 1)
   1. Add antibiotics to the LB medium at final concentrations of kanamycin (40 μg/mL) and chloramphenicol (10 μg/mL).
   2. Aliquot 5 mL of LB/Kan40/CM10 medium into sterile test tubes with caps.
   3. Inoculate E. coli strains from freezer stocks: E. coli culture expressing LuxR/HapR and pJV064 plasmid (LuxR+)^9,15; E. coli empty vector control culture and pJV064 plasmid (LuxR-)^9,15.
   4. Shake the culture overnight at 275 rpm at 30 °C for 16-18 h.
3. Assay of thiophenesulfonamides (Day 2)
   1. Weigh out ~30 mg of the compound. Resuspend to 100 mM in DMSO and vortex to mix. Next, prepare a 10 mM stock.
   2. Mix 20 μL of the 100 mM stock with 180 μL of DMSO to dilute it (1:10) to yield a 10 mM working stock. Vortex to mix.
   3. Back-dilute LuxR+ culture and LuxR- cultures (1:100) in 10 mL of LB/Kan/CM media into 15 mL conical tubes and vortex briefly to mix.
   4. Use a black-well, clear-bottomed, sterile 96-well plate for the assay.
   5. Prepare a dilution series for all columns. A diagram of the 4-fold dilution series is included as a reference (Figure 4B).
      NOTE: A digital multi-channel pipet is recommended for this process. This method is for a 4-fold dilution series (1:4). However, different dilution series may be used (e.g., 1:10, 1:5).
      1. Pipet 200 µL of the LuxR+ culture mix into a 96-well plate in row A, 1-6 (Figure 4B; green).
      2. Pipet 200 µL of the LuxR- culture mix into a 96-well plate in row A, 7-12 (Figure 4B; orange).
      3. Pipet 150 µL of the LuxR+ culture mix into a 96-well plate in rows B-E, 1-6 (Figure 4B; green).
      4. Pipet 150 µL of the LuxR- culture mix into a 96-well plate in rows B-E, 7-12 (Figure 4B; orange).
      5. Add 2 µL of the compound or DMSO to each well of row A according to Figure 4B.
      6. For every column, pipet up and down 3 times, then transfer 50 µL from row A into row B, in the same column.
      7. Repeat the mixing and pipetting for rows B, C, D, E. After mixing row E, remove 50 µL and put in waste. In the end, one should have 150 μL in each well.
   6. Cover the plate with microporous tape.
   7. Incubate the plate by shaking at 275 rpm at 30 °C overnight for 16-18 h.
   8. Measure the fluorescence and optical density of the assay plates (Day 3).
      1. Remove the tape carefully, and do not spill liquid out of the wells.
      2. On a plate reader, read the optical density at 600 nm (OD₆₀₀), GFP, and mCherry.
         NOTE: A set gain for both fluorescence channels is recommended to enable comparisons between assays.
   9. Record data as normalized fluorescence per cell: GFP/OD₆₀₀ and mCherry/OD₆₀₀.
   10. Plot the data, as shown in Figure 5, to illustrate the results for the DMSO control in comparison to the test samples.

4. Washing the black assay plates for re-use

1. Soak the plates in bleach.
   1. Label a 1 L plastic beaker as "biological waste". Dump liquid from plates into this waste container (carefully clean up any drips with 70% EtOH). Rinse the plates with DI water from the squirt bottle over the waste and dump it into it.
   2. Label another 1 L plastic beaker as "30% bleach". Put plates/lids in this container and cover with 30% bleach. Wrap the top with a plastic sheet and weigh the plates with a tip box or something similar. Let sit submerged in the bleach solution overnight.
2. Rinse and soak in ethanol.
   1. Thoroughly rinse the plates with DI water into the sink so that no bleach remains. Dump out any remaining water from wells into the sink.
   2. Add 70% EtOH to all wells and lids using a spray bottle. Let the plates sit upright with the lid on top but askew so that the EtOH can evaporate, but nothing falls into the wells. Let the plate sit overnight.
3. Allow the plates to dry.
   1. Dump out any remaining EtOH.
   2. Flip over the plate onto a paper towel and let the rest of the EtOH evaporate. Leave the plates this way in a fume hood. The plates will be ready for the next use.

Representative Results

As representative results, data is included from three thiophenesulfonamide compounds synthesized by undergraduate students for compounds 1A, 2B, and 3B (Figure 5A-C; described in detail in Newman et al.⁹). Each compound was tested in the E. coli strain expressing V. campbellii LuxR and using the pJV064 reporter plasmid. The normalized fluorescence per cell is shown for each assay. The assay was performed with a 1:10 dilution series to assay concentrations 1 µM, 10 µM, and 100 µM of each compound compared to the solvent control. Compounds 1A and 3B were each inhibitory though to different extents: 1A only exhibited inhibition of LuxR at 100 µM, as evident by the loss of GFP expression and increase in mCherry expression. Conversely, 3B inhibited LuxR activity at all three tested concentrations. Compound 2B did not have any activity; thus, all concentrations tested appeared similar to those of the DMSO solvent control. A minimum of three biological replicates of each assay is recommended to perform statistics and determine the data distribution and error.

Figure 1: LuxR/HapR proteins are the master QS regulators in Vibrios. (A) Schematic of the generic QS circuit in Vibrio species. Membrane-bound histidine kinase receptors bind autoinducers, which leads to the production of the master QS transcriptional regulator LuxR/HapR. Group behaviors are activated, which typically include numerous virulence factors that contribute to vibriosis disease. Thiophenesulfonamide inhibitors such as compound PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H-pyrazole) bind to LuxR/HapR and block its function, thus eliminating group behaviors. (B) Pymol rendering of a docked structure: V. vulnificus SmcR protein in which PTSP has been modeled into the binding pocket using Webina. Please click here to view a larger version of this figure.

Figure 2: Synthesis of thiophenesulfonamides. (A) General scheme for the synthesis of thiophenesulfonamide compounds. (B) Scheme for the synthesis of PTSP. Please click here to view a larger version of this figure.

Figure 3: Modeling of PTSP in SmcR. (A) Output from Webina that shows PTSP docked into the ligand binding pocket of SmcR from Vibrio vulnificus. (B) A figure from a student report showing a Pymol rendering of the same interaction. Please click here to view a larger version of this figure.

Figure 4: E. coli screen for thiophenesulfonamide inhibitors of LuxR/HapR. (A) Diagram of the E. coli red-green screen reporter plasmid and LuxR/HapR expression plasmid. (B) Diagram of the 96-well assay plate setup for screening thiophenesulfonamide compounds. Please click here to view a larger version of this figure.

Figure 5: Student data assessing three thiophenesulfonamides in the E. coli assay. Three compounds (A-C) were synthesized and assayed as described in this protocol. The normalized GFP and mCherry fluorescence are shown for all three compounds compared to the DMSO solvent control at equal volume. These data are from a single biological assay for each compound. Please click here to view a larger version of this figure.

Figure 6: Moderate LuxR inhibitors with unexplored structural motifs. Please click here to view a larger version of this figure.

Supplementary Figure 1: ASURE lab two-semester program. Please click here to download this File.

Discussion

This CURE was originally developed as an abbreviated two-stage, three-week protocol (design/synthesis and assay) and was implemented in five semesters as part of an upper-level organic laboratory course⁸. Since the original report, the computer modeling module was added, and the E. coli assay was optimized for novice researchers. The resulting three-stage, two-semester protocol has been implemented three times as part of Indiana University's Arts and Sciences Undergraduate Research Experience (ASURE) program, which is taken by students in their first and second years. The overall structure of the course changes year to year as research goals evolve; an example schedule is outlined in Supplementary Figure 1.

Procedures for one specific compound (PTSP) and two target proteins (LuxR and SmcR) are described herein; however, the value and longevity of this CURE lie within its inherent flexibility. More than 100 compounds have been synthesized in the context of these courses with various levels of activity or inactivity in the E. coli assay. This synthetic library does not scratch the surface of the potential chemical space yet to be explored. Most of the synthetic library consists of PTSP derivatives with variously substituted pyrazole and thiophene groups, leaving much room for investigation. For example, the benzenesulfonamide (1E), amide (3F), and pyrrolidine (P0074 H4) derivatives shown in Figure 6 have moderate activity against LuxR and SmcR, and none of these scaffolds have been explored in depth¹⁰. In addition to the unexplored chemical space, there are several LuxR homologs for which there is no effective inhibitor¹⁰. A variation of this CURE could be designed around the exploration of chemical space or, around one of the less sensitive LuxR homologs, or with a combination of these research goals.

This set of protocols is a unique combination of chemical synthesis, computer modeling, and microbiology in a single course that enables students to emulate the medicinal chemistry process for drug design. This CURE model has inherent flexibility and can be implemented in many different ways, or it can serve as a model for a laboratory course designed around small-scale drug design in any target.

Materials

Name	Company	Catalog Number	Comments
2-thiophensulfonyl chloride	Ambeed	A258464
3-Phenyl-1H-pyrazole	Ambeed	A104401	98%
96-well clear bottom black plates	USA Scientific	5665-5090Q	96-well polystyrene uClear black TC plate with lid, clear flat bottom, sterile, 8/sleeve, 32/case
Autodock Tools			http://mgltools.scripps.edu/downloads
Autodock Vina			https://vina.scripps.edu
Chloramphenicol
DMSO
ethyl acetate	Fisher Scientific	AA31344M4	Reagent grade
hexanes	Fisher Scientific	H291
Kanamycin
magnesium sulfate	Fisher Scientific	M65-500	Anhydrous
Microporous Film	USA Scientific	2920-1010	Microporous Film, -20degC to +80degC, 50/box, Sterilized
molview			molview.org
NaCl
Protein Databank			https://www.rcsb.org/
Pymol			https://pymol.org/2/
Qualitative filter paper	Fisher Scientific	09-805-342	Cytiva Whatman™ Qualitative Filter Paper: Grade 1 Circles, 47 mm
Silica gel	Sorbtech	30930M-25	Silica Gel, Standard Grade, 60A, 40-63um (230 x 400 mesh)
Sodium hydride	Millipore Sigma	452912	60 % dispersion in mineral oil
Tetrahydrofuran	Fisher Scientific	MTX02847	Tetrahydrofuran, anhydrous, 99.9%, ACS Grade, DriSolv
TLC Plates	Sorbtech	1634067	Silica gel TLC plates, aluminum backed
Tryptone
webina			https://durrantlab.pitt.edu/webina/
Yeast Extract