Team:BYU Provo/Notebook/Cholera - Enzymes/March-April

Overview

March-April

May-June

July-August

September-October

One of the problems that we might have after cloning it into the E coli strain is whether or not the sRNA genes will clone in successfully. We will be able to determine whether or not they do when we place the E coli in a site with HCD and LCD of cholera. If the rfp is activated in both then we will know that the sRNA’s weren’t transferred in succesfully and we will need to  figure out a different approach.

Today we were able to start a map of the plasmid. In all we will have an estimated cloning site of around 1100 bp’s. Clarise started working on the primer details to make sure we have enough primers to cover the entire gene during sequencing. So far it looks like the only gene that we need to insert another primer in is the first one which is LuxPQ I worked on the plasmid design. We also requested a strain of Cholerae from Dr. Robison’s lab and we will have that on Wednesday to start work with. Wednesday we will need to complete the process in order to submit it into the sequencing center by Thursday.

CH 3/18/13

See Nathan’s entry above.

MS - 3/18/13

I was in California for my sister's mission farewell this weekend and wasn't able to make it back in time for our lab so I worked on starting to compile the map of our plasmid. I put all the nucleotide sequences for the different genes in the order that they will be in the plasmid, but am unsure about how they will be linked specifically. I put together what I had and emailed it to Nathan and Clarice so that they can use it during the lab today.

March 18th, 2013- Whitney Hoopes

Researched and read articles pertaining to biofilm degradation: (Also reviewed quorum sensing articles)

Alina Nakhamchik, C. W., and Dean A. Rowe-Magnus (2008). "Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus." Applied and Environmental Microbiology 74(13): 4199-4209.

Anneleen Cornelissen, P.-J. C., Jeroen T'Syen, Helena Van Praet, Jean-Paul Noben, Olga V. Shaburova, Victor N. Krylov, Guido Volckaert, Rob Lavigne (2011). "The T7-related Pseudomonas putida phage O15 displays virion-associated biofilm degradation properties." PLoS ONE 6(4): e18597.

Bassler, W.-L. N. a. B. L. (2009). "Bacterial Quorum-Sensing Network Architectures." Annual Reviews 43: 197-222.

Christopher M. Waters, W. L., Joshua D. Rabinowitz and Bonnie L. Bassler (2008). "Quorum sensing controls biofilm formation in Vibrio cholerae through modulation of cycli di-GMP levels and repression of vpsT." Journal of Bacteriology 190(7): 2527-2536.

Cynthia Wu, J. Y. L., Gerald G. Fuller, and Lynette Cegelski (2013). "Disruption of Escherichia coli amyloid-integrated biofilm formation at the air-liquid interface by a polysorbate surfactant." Langmuir 29: 920-926.

D.H. Dusane, J. K. R., A.R. Kumar, Y.V. Nancharaiah, V.P. Venugopalan and S.S. Zinjarde (2008). "Disruption of fungal and bacterial biofilms by lauroyl glucose." Letters in Applied Microbiology 47: 374-379.

Jun Zhu, M. B. M., Russell E. Vance, Michelle Dzlejman, Bonnie L. Bassler and John J. Mekalanos (2002). "Quorum-sensing regulators control virulence gene expression in Vibrio cholerae." PNAS 99(5): 3129-3134.

Yildiz, J. C. N. F. a. F. H. (2007). "The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae." Journal of Bacteriology 189(6): 2319-2330.

Yildiz, J. C. N. F. a. F. H. (2008). "Interplay between cyclic AMP-cyclic AMP receptor protein and cyclic di-GMP signaling in Vibrio cholerae biofilm formation." Journal of Bacteriology 190(20): 6646-6659.

-Begin growing cholera biofilms to test last years constructs -Sequence plasmid constructs on Wednesday

NRS 3-20-2013 Last night Kendall told me that there was another iGEM project from the Calgary team that used Quorum sensing extensively for detection and destruction. So today we informed Dr. Grose and did some more research into it. Since 2008 there has been at least one project every year that has used it in some sense. Most used V. fischeri and only one used V. cholerae but it wasn’t to the extent that we are aiming our project at. We decided to wait until Friday to decide whether or not we will continue with this project. One of the benefits of keeping with this project is the potential real world application that it could have for the treatment of water.

CH 3/20/13

It turns out that last year’s team had one step to go before the complete construct was made with all quorum sensing genes. We will sequence through what is already made and then finish what they started.

The problem was brought up that another team did a very similar project in 2008. I think the biggest question for us now is if our project is different enough from what they did. They replicated the quorum sensing system found in V. harveyi in E.coli. This is very similar to our detection half of the Cholera project. The trick is if we have a good use for transferring the V. cholerae QS into E.coli. Our project can’t be centered around this. We need some kind of application.

Whitney Hoopes March 20th Overview of E. coli transformation protocol:

Eppendorfs of DH5alpha cells---thaw on ice Mix 2-5ul of plasmid and keep on ice for 20-30minutes Heat shock for 1 min @ 42 Degrees Celsius Cool eppendorf on ice for 2-5 minutes Add 0.5 mL LB and incubate at 37 Degrees Celsius for 30 minutes (select for AMP) Plate on selective media (LB-AMP) Phage Titer Protocol

Plan: -Need to set up plasmids for sequencing -Grow a Cholera biofilm -Gram-stain of Cholera to check

MS - 3/20/13

I was sick with a flu bug that I picked up in California and was unable to go to lab today. Instead, I looked up and read several recent research articles on the quorum sensing system in Vibrio cholerae in order to understand it better. The references for the articles I read are:

Hoch, J. Two-Component and Phosphorelay Signal Transduction. Curr. Opin. Microbiol. 2000, 3, 165–170.

Miller, M.; Skorupski, K.; Lenz, D.; Taylor, R.; Bassler, B. Parallel Quorum Sensing Systems Converge to Regulate Virulence in Vibrio cholerae. Cell 2002, 110, 303–314.

Waters, C.; Bassler, B. Quorum Sensing: Cell-to-Cell Communication in Bacteria. Annu. Rev. Cell Dev. Biol. 2005, 21, 319–446.

Zhu, J.; Miller, M.; Vance, R.; Dziejman, M.; Bassler, B.; Mekalanos, J. Quorum-Sensing Regulators Control Virulence Gene Expression in Vibrio cholerae. Proc. Natl. Acad. Sci. 2002, 99, 3129–3134.

MS - 3/22/13

It came to our attention that several previous iGem teams have done work with quorum sensing systems in the past. One team was able to transfer the quorum sensing sytem of Vibrio harveyi into E. coli. Also, at least one team, the St. Andrews 2010 team, has worked on cholera detection, but were unsuccessful in their attempts. We met together as an entire team and discussed this issue to decide whether we would continue with the cholera project or not. After discussion, we decided that although work on this has already been done, the scope of what we are attempting with both detecting and destroying cholera biofilms is still unique so we will continue with the cholera project. However, we will change the approach of our cholera project to incorporate phage into the destroy part of the project. This will make our project even more novel and will bring the two projects closer together so that our overall presentation will be more coherent.

Due to the restructuring of our cholera approach we have decided to change the subgroups of our cholera team and our focuses. The two destroy teams will be the phage team and the enzyme team. I am on the enzyme team and we decided that we will each choose three research articles to read in detail this weekend. The articles I am going to read are:

Thallinger, B.; Prasetyo, E.; Nyanhongo, G.; Guebitz, G. Antimicrobial Enzymes: An Emerging Strategy to Fight Microbes and Microbial Biofilms. Biotechnol. J. 2013, 8, 97-109.

Lamppa, J.; Griswold, K. Alginate Lyase Exhibits Catalysis-Independent Biofilm Dispersion and Antibiotic Synergy. Antimicrob. Agents Chemother. 2013, 57, 137-145.

Molobela, I.; Cloete, T.; Beukes, M. Protease and Amylase Enzymes for Biofilm Removal and Degradation of Extracellular Polymeric Substances (EPS) Produced by Pseudomonas fluorescens Bacteria. Afr. J. Microbiol. Res. 2010, 4, 1515-1524.

CH 3/22/13

After our in-class discussion we decided to keep moving forward with the Cholera project. Turns out that the other team cloned in the QS genes from V. harveyi which has enough differences from the cholera system to make it worth it to keep moving forward. I am now working with Kelton and Kendal on looking more into biofilm degradation using phage. We are going to do a two pronged approach. One side will involved engineering a known cholera phage with modifications that will degrade biofilm. The other side is a search for cholera associated phage. My objectives for the next bit of time: -Research where cholera is usually located globally and nationwide (maybe there is a researcher in the US who could send us a sample of contaminated water) -Research the nature of cholera biofilm -Research general characteristics of phage/biofilm interactions using papers found in table 3 of the Review -Do the sequencing analysis for the construct that was made by the old iGEM team (need lists of primers, sequence, and general gene length)

Whitney Hoopes 3/22/13 Want to clone in CqS---specific (intra receptor)---signals to LuxO

detection/elimination novel EXDP associates with the tail fibers of the phage

2 cholera phage reported

Options: 1. engineer a phage 2. go phage hunting

Focus on the elimination system Phage degradation genes

Decide/Research papers to read for enzyme degradation Ilana Kolodkin-Gal, S. C., Liraz Chai, Thomas Bottcher, Roberto Kolter, Jon Clardy, and Richard Losick (2012). "A self-produced trigger for biofilm disassembly that targets exopolysaccharide." Cell 149: 684-692.

N. Ramasubbu, L. M. T., C. Ragunath and J.B. Kaplan (2005). "Structural analysis of Dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans." Journal of Molecular Biology 349: 475-486.

Anneleen Cornelissen, P.-J. C., Victor N. Krylov, Jean-Paul Noben, Guido Volckaert, Rob Lavigne (2012). "Identification of EPS-degrading activity within the tail spikes of the novel Pseudomonas putida phage AF." Virology 434: 251-256.

Dev K. Ranjit, J. L. E. a. K. W. B. (2011). "Staphylococcus aureus CidA and LrgA proteins exhibit holin-like properties." Journal of Bacteriology 193(10): 2468-2476.

MS - 3/23/13

The following are my summary notes from the above articles: Lamppa, J.; Griswold, K. Alginate Lyase Exhibits Catalysis-Independent Biofilm Dispersion and Antibiotic Synergy. Antimicrob. Agents Chemother. 2013, 57, 137-145.

P. aruginosa infects the airways of cystic fibrosis patients and forms a mucoid biofilm. The major component of the biofilm is alginate. Alginate is a copolymer of (1,4)-linked beta-D-mannuronic acid and alpha-L-guluronic acid. Two alginate lyases were studied: A1-III and AlgL. When tested individually, each showed slight biofilm degradation (25-30%) degradation. However, when either was used in conjunction with tobramycin there was 100% biofilm degradation. This was tested on forms of the bacteria that both do and don't create alginate, thus the biofilm degradation was not linked to the alginate lyase function specifically. The same results occurred whether or not they used the intact lyase protein, an inactive mutant, or the individual amino acids. They postulate that the lyase enzyme is used as a food source, the presence of which induces a natural partial breakdown of the biofilm as bacteria spread to grow. This spreading raises the efficiency of the tobramycin and allows for 100% biofilm degradation. I wonder if we could produce the environmental triggers that cause cholera biofilms to break apart in the intestines, we might be able to do something similar or if we

Molobela, I.; Cloete, T.; Beukes, M. Protease and Amylase Enzymes for Biofilm Removal and Degradation of Extracellular Polymeric Substances (EPS) Produced by Pseudomonas fluorescens Bacteria. Afr. J. Microbiol. Res. 2010, 4, 1515-1524.

One major problem of degrading biofilms is that the effectiveness of an enzyme to degrade a biofilm depends upon the specific composition of the biofilm and its extracellular polymeric substance. Synthetic polysaccharides have been used in the past to degrade the biofilms of several bacteria, but are not effective with all biofilms. They grew Pseudomonas fluorescens biofilms, then extracted the EPS to determine its carbohydrate and protein composition. A series of commercial enzymes were tested with the proteases Savinase and Everlase the most effective in reducing protein concentration within the EPS. They postulate that proteases will be most effective against EPS with a higher protein concentration and amylase enzymes will be most effective against EPS with a higher carbohydrate concentration. However, they also believe that the relative concentrations of proteins and carbohydrates depends upon the growth environment of the biofilm and can change from location to location, even for the same species of bacteria.

Thallinger, B.; Prasetyo, E.; Nyanhongo, G.; Guebitz, G. Antimicrobial Enzymes: An Emerging Strategy to Fight Microbes and Microbial Biofilms. Biotechnol. J. 2013, 8, 97-109. This is a review of several enzymes shown to be effective in degrading biofilms and/or destroying bacteria There are two main types of enzymes used to degrade biofilms: proteases and amylases. This is because biofilms are primarily a mixture of proteins and polysaccharides. The list of effective enzymes in this review could help us supplement our list of potential enzymes to use

NRS 3/25/13

Today we need to start several things. First is sequencing the plasmid the team made last year. Second is growing the biofilms at room temperature, 30 degrees C and 37 C to see which results in the best formation of biofilm.

One article I read was about different plants that broke down biofilms http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591221/ This review lists and talks about several categories of plant based products that degrade the biofilm

Whitney Hoopes 3/25/13 Set up O/Ns of cholera in 4mL LB at room temperature, 30 Celsius and 37 Celsius -Not shaking with control blank Sequencing 57 (f) with 12 (reverse) with pIG 85 and 86 -4 samples to be sequenced Streaked cholera on LB plate in 30 degree incubator

MS - 3/25/13

We presented our current plans as a team so that we are all on the same page. For our enzyme approach, last year's team only cloned in two enzymes to test for biofilm degradation. We took samples of those enzymes and prepared them for sequencing. For this we used the pIB85 Forward, pIB Reverse, pIB86 Forward, and pIB86 Reverse primers for the (Cytr?) and (Ama?) enzymes. For each sample 2 μL of plasmid and 1 μL of primer was used. The prepared samples were sent to be sequenced for us to determine if our plasmids are ready to go.

Possible additional enzymes to test: Subtilisins - produced by Bacillus sp., are serine proteases that hydrolyze adhesins. Shown to remove biofilms of several bacteria, but not yet tested on cholera specifically. Alpha-Amylase - inhibits formation of, and hydrolyzes existing biofilms in S. aureus. Can be used in conjunction with proteases to increase biofilm degradation. Dispersin B - produced by Actinobacillus actinomycetemcomitans. Combining with proteases, DNAases, and glycolytic enzymes enhances biofilm removal.

The following are articles I am going to try to read before Wed: Sumantha, A., Larroche, C., Pandey, A., Microbiology and industrial biotechnology of food-grade proteases: a perspective. Food Technol. Biotechnol. 2006, 44, 211–220.

Darouiche, R.O., Mansouri, M.D., Gawande, P.V., Madhyastha, S., Antimicrobial and efficacy of triclosan and DispersinB¨ combination. J. Antimicrob. Chemother. 2009, 64, 88–93.

Esperanza Torres, C., Lenon, G., Craperi, D., Wilting, R. et. al., Enzymatic treatment for preventing biofilm formation in the paper industry. Appl. Microbiol. Biotechnol. 2011, 92, 95–103.

MS - 3/27/13

Today we nailed down more of what we plan to do. We are currently growing V. cholerae cultures at 37 °C, 30 °C, and room temperature. We started growing our cultures on Monday and do not have any biofilms yet. Once our biofilm cultures have grown we are going to focus our enzyme tests on three enzymes at first: Amylase (AmyA) We have a plasmid from last year that we sequenced and is ready to use once the cholera cultures are ready. Subtilisin Savinase - from Bacillus lentus We have the primers from last year and need to get the genes so that we can start cloning them into E. coli Dispersin B - from Actinobacillus pleuropneumoniae We have the primers from last year and need to get the genes so that we can start cloning them into E. coli.

Per Dr. Grose's suggestion I emailed Dr. Rich Robison to ask if he has Bacillus lentus that we could use to get a DNA template for the gene we want. Once we have the bacteria we can start cloning the targeted genes into E. coli to prepare to test on our cholera biofilms.

On Monday, we are going to give a presentation on where we are at in our research and our specific experimental design. We divided the presentation between the cholera phage and enzyme groups. We (the enzyme group) will do the general overview and each of us will present one of our three enzymes, I have the Subtilisin Savinase enzyme. Also, we are each going to find four research articles to read about the cholera biofilm to find out what's already known about it.

3/27/13 Whitney Hoopes Cholera papers I will study and present on Monday:

Jason B. Harris, R. C. L., Firdausi Qadri, Edward T. Ryan, Stephen B. Calderwood (2012). "Cholera." The Lancet 379(9835): 2466-2476.

Luanne Hall-Stoodley, P. S. (2002). "Developmental regulation of microbial films." Current Opinion in Biotechnology 13(3): 228-233.

Shuyang Sun, S. K., Diane McDougald (2013). "Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists." PLoS ONE 8(2): e56338.

Nicholas J. Shikuma, K. R. D., Jiunn N.C. Fong and Fitnat H. Yildiz (2012). "The transcriptional regulator, CosR, controls compatible solute biosynthesis and transport, motility and biofilm formation in Vibrio cholerae." Environmental Microbiology.

M. Kamruzzaman, S. M. N. U., D. Ewen Cameron, Stephen B. Calderwood, G. Balakrish Nair, John J. Mekalanos, and Shah M. Faruque (2010). "Quorum-regulated biofilms enhance the development of conditionally viable, environmental Vibrio cholerae." PNAS 107(4): 1588-1593.

Katrina L. Van Dellen, P. I. W. (2006). "The Vibrio cholerae biofilm: A target for novel therapies to prevent and treat cholera." Drug Discovery Today: Disease Mechanisms 3(2).

-Sequenced plasmids 85F, 85R, 86F, 86R -Appear to be correct Cytr and AmyA genes cloned in to the E. coli plasmid -Focus on studying AmyA gene for presentation -Started Cholera overnights in 30 degrees and 37 degrees incubators. Left overnight in the shakers to better disperse the cholera growth -Moved out and onto the shelf 3/28/13 -Our group is in charge of presenting a general introduction for our iGEM project -Include the basic biofilm information

NRS 3/27/2013 Today we discussed a little bit more what we have planned to do. I am reading the following articles for our presentation on monday.

Mudrack, Benjamin Tamayo Rita (2012) “The Vibrio cholerae Pst2 Phosphate Transport System Is Upregulated in Biofilms and Contributes to Biofilm-Induced Hyperinfectivity” “ Therefore, the physiological state of individual biofilm-associated cells is responsible for the observed hyperinfectivity.” Does this mean that even if we break down the bio-film that v. cholerae would still be infectious?

Tapas Patra, Hemanta Koley, Thandavaryan Ramamurthy, Asoke C. Ghose, Ranjan K. Nandy. “The Entner-Doudoroff Pathway Is Obligatory for Gluconate Utilization and Contributes to the Pathogenicity of Vibrio cholerae” The Entner -Doudoroff pathway plays a role is the catabolism of sugars It can be turned on or off depending on which phase the V. cholerae is in. Deactivation of the ED pathway positively affects biofilm formation.

Valeru SP, Wai SN, Saeed A, Sandström G, Abd H. “ToxR of Vibrio cholerae affects biofilm, rugosity and survival withAcanthamoeba castellanii”

MS - 3/27/13

Today we nailed down more of what we plan to do. We are currently growing V. cholerae cultures at 37 °C, 30 °C, and room temperature. We started growing our cultures on Monday and do not have any biofilms yet. Once our biofilm cultures have grown we are going to focus our enzyme tests on three enzymes at first: Amylase (AmyA) We have a plasmid from last year that we sequenced and is ready to use once the cholera cultures are ready. Subtilisin Savinase - from Bacillus lentus We have the primers from last year and need to get the genes so that we can start cloning them into E. coli Dispersin B - from Actinobacillus pleuropneumoniae We have the primers from last year and need to get the genes so that we can start cloning them into E. coli.

Per Dr. Grose's suggestion I emailed Dr. Rich Robison to ask if he has Bacillus lentus that we could use to get a DNA template for the gene we want. Once we have the bacteria we can start cloning the targeted genes into E. coli to prepare to test on our cholera biofilms.

On Monday, we are going to give a presentation on where we are at in our research and our specific experimental design. We divided the presentation between the cholera phage and enzyme groups. We (the enzyme group) will do the general overview and each of us will present one of our three enzymes, I have the Subtilisin Savinase enzyme. Also, we are each going to find four research articles to read about the cholera biofilm to find out what's already known about it.

MS - 3/29/13

I received an email back from Dr. Robison, he doesn’t have either of the bacteria that we are looking for. Whitney and I went down to the department secretaries and they agreed to forward an email from us to all the faculty in the department to see if we can locate our bacteria. I sent them the email and we are waiting for a response.

The following are the papers that I read about biofilms in V. cholerae.

Sun, S.; Kjelleberg, S.; McDougald, D. Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists. PLoS One 2013, 8, PMC3575383.

Kalpana, B.; Aarthy, S.; Pandian, S. Antibiofilm Activity of alpha-Amylase from Bacillus subtilis S8-18 Against Biofilm Forming Human Bacterial Pathogens. Appl. Biochem. Biotechnol. 2012, 167, 1778-1794.

Augustine, N.; Peter, W.; Kerkar, S.; Thomas, S. Arctic Actinomycetes as Potential Inhibitors of Vibrio cholerae Biofilm. Curr. Microbiol. 2012, 64, 338-342.

He, H.; Cooper, J.; Mishra, A.; Raskin, D. Stringent Response Regulation of Biofilm Formation in Vibrio cholerae. J. Bacteriol. 2012, 194, 2962-2972.

Here are notes of what I learned from these papers about biofilms in V. cholerae: Biofilm formation regulated by a variety of signals, including salinity, bile, calcium, and phosphate. Genes for EPS formation are found in two vps (Vibrio exopolysaccharide) operons, one consisting of vpsU and vpaA to -K, and the other consisting of vpsL to -Q. There are two known transcriptional activators of the vps operons, VpsR and VpsT. vpsR mutants are completely defective in biofilm formation Expression of vpsR and vpsT is regulated by the expression of hapR. At high cell density, hapR is expressed, and vpsR and vpsT are repressed. Stringent response induces biofilm formation, particularly when V. cholerae is forming biofilms in aquatic environments, where there may be low nutrient availability. Stringent response is induced when there is an increased concentration of the second messengers pppGpp (guanosine 3=-diphosphate 5=triphosphate) and ppGpp [guanosine 3=5=-bis(diphosphate)], together termed (p)ppGpp. An increased concentration of (p)ppGpp causes significant changes in gene expression that result in cessation of growth and induction of specific stress responses. Deletion of (p)ppGpp synthases led to loss of the ability to form biofilm and a decrease in expression of vpsR and vpsT. In stringent response-induced cultures, vpsR expression increased 2.5-fold and vpsT expression increased 4-fold.

March 29th, 2013 Whitney Hoopes Read articles (provided in 3/27) and worked on presentation Checked Cholera O/Ns----still no visible growth

3/29 NRS

I still am researching the articles listed under 3/27 and we are working on our presentation for Monday.