Cholera - Detection

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March

3/15/13

KP 3/15/13 Today as we compared our individual findings on methods to destroy the biofilm on cholera, we realized that there are many different approaches to take. We can try to stop biofilm production by disrupting the quorum sensing, destroy the internal mechanism that makes the biofilm, or we can attack the biofilm from the outside. We have decided to focus on that last approach. Our goal now, is to find an enzyme or chemical of some kind and then see if it will break down the biofilm of cholera. There are plenty of enzymes already discovered that break down biofilms on other bacteria, but we cannot find anything specific to cholera. We will probably have to experiment with different proven methods for other bacteria and see what effects it has on cholera. We can also possibly incorporate phage as a deliverer of a substance that breaks down cholera biofilm, but right now we are in search of that enzyme.

KK 3/15/13 We reported on our investigation of various genes and enzymes that last year’s iGEM team had identified as candidates to disrupt the biofilm. Their approaches appear very disjointed: some proteins were meant to directly degrade the biofilm, some were meant to hydrolyze the QS autoinducer molecule, some (appear) to interfere with transcription and translation of HapR related genes. While the advantage to their multi-front approach is that perhaps one of their various technique could work, the disadvantage of being spread too thin I believes overrides the advantage. So, we’ll be homing in on only one approached: direct removal of the biofilm. Dr. Grose answered a question that had come up as I researched. I learned that two serogroups of Cholera are pathogenic to humans: O1 and O139. I wondered if it were important that the species of cholera that we were to work with be of those two serotypes. Dr. Grose explained that bacteria are grouped serotyped based upon how our immune system reacts to them. It isn’t important what serotype cholera is, because any type will make a biofilm. Also, if the biofilm is removed, any type can be destroyed by stomach acid. Questions to Research: What is the composition of the biofilm in Cholera? Exopolysaccharides (EPS) are a principal building block. EPS in most species has a negative charge and is (thus not surprisingly) chlorine resistant. What other components are there? Has anything successfully degraded its biofilm? We know of multiple papers that document biofilm reduction in seemingly every species BUT cholera, but we have failed to find any that is cholera-specific.

March 15th- Whitney Hoopes -Compared research articles/data gathered for ideas on how to destroy the cholera biofilm -Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Use quorum sensing to trigger transcription of genes that turn on and produce enzyme transcripts to degrade the biofilm? -Reviewed research papers and reviews to gather ideas and plan what genes to clone in -Discussed genes we researched: Dispersin (DspB) 106/107 Aiia 109/110---B. subtilis CytR 111/112 Deoxyribonuclease 113/114---B. subtilis Subtilisin sowirlane 115/116---B. subtilis Apple favonoid 117/118 Nuclease NuCB 119/120 Dnase 1 121/122 Amylase (AmyA) 123/124 Biofilm targets Holin endolysin 129/130 Anti-LPS 125/126 (bacteria target) Out of biofilm ChapK

3/18/13

KP Mon. March 18 Cholera Destroy Plan of attack- Get Vibrio Cholerae Grow Biofilm Find constructs from last year Test constructs against biofilm Construct our own biofilm destroyer to test out.

Cholera Detect There is already a lot done from last year, so we need to test the constructs from last year. The first step is sequencing and then getting it into E. Coli. Just like the destroy group, we first need to start growing some Cholera.

Big phage Need to do more research on what has already been done. One possible question is how to get a plasmid to form with medicine or something inside.

Small Phage They are trying to decide which phage to use. They are going to grow phage and see if any mutations produce a smaller phage. They need to come up with a screening method.

Here is what we need to do right now. We went to Dr. Robison’s lab and asked for a sample of cholera. We will return on Wednesday to pick it up. It is important that we can get the cholera to grow and produce biofilm. Next, we are looking through the work that the iGEM team did last year. We want to try some of their constructs on the biofilm to see if they actually work. We will also try some of our own ideas.

KK March 18th,

Our priority list right now is: 1) Get cholera growing a biofilm, and 2) Test existing constructs from last year’s iGEM.

We talked with Annette, Dr. Robison’s lab manager, and she promised us she would have a streak of cholera available for us by Wednesday. As soon as we get Cholera, we’re going to form a plan with Dr. Grose for how we can store it so as to not need to constantly ask Dr. Robison’s lab for cholera. On Wednesday we’ll begin to see if we can get cholera to grow biofilms.

Today I looked over existing plasmid constructs. Apparently iGEMers from last year did clone many genes into plasmids but the plasmids aren’t yet in the registry. Since the plasmids have not been sequenced, that will be another priority on Wednesday.

We also learned that Calgary in 2008 created “Champion,” a bacteria that sensed AHL and AI-2. They used colicin to succesfully eliminate “Bad Guy 1” (which I think is so tacky, and I struggled to determine what the heck “Bad Guy” is but I think it’s Vibrio Fischeri). Their quorum-sensing construct worked … they did NOT work with cholera, but in their presentation video they make reference to the potential usefulness of their system with cholera.

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 (waiting until we have final decision on project)

3/20/13

KK Today is March 20th, two thousand thirteen

We were thrilled to get out plate of cholera today! Then we happened upon about 5 iGEM projects from previous years that involve quorum sensing and/or cholera. We were on the verge of beginning our lab work but our paradigm was suddenly shifted a few steps backward: we needed (and still need) to decide if we should continue working on our cholera project. Calgary’s 2008 iGEM team successfully cloned the quorum-sensing circuit into E.Coli and used it to detect and destroy E.Coli that was expressing AHL - whose homologs appears to be autoinducers of V. Harveyi and V.Fischeri. St. Andrew’s iGEM team faced off against cholera specifically, attempted to render it avirulent through generating high levels of cholera’s autoinducer CAI-1, but they did not tame cholera. They only managed to get the CAI-1 autoinducer to work in E.Coli.

We got our cholera from Annette, Dr. Robison’s lab manager. She plated it on Columbia media, and it had been sitting at 37 degrees C for 24 hours when she gave it to us. We don’t know the exact assay for growing biofilm in cholera, but some protocols suggest LB broth. Our plan is to use LB broth and let cholera grow (NOT shaking it, so that biofilm may grow freely).

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

KP March 20th

Here are a few good papers on using phage to destroy biofilm.

https://www.novapublishers.com/catalog/product_info.php?products_id=21940

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797112/

How Phages Kill Biofilm

http://www.biomedcentral.com/1472-6750/8/79

Phage That Are Capable of Destroying Biofilms

Bacteriophage Phi S1 infection of Pseudomonas fluorescens planktonic cells versus biofilms. Pseudomonas fluorescens biofilms subjected to phage phiIBB-PF7A. Lytic Activity of Recombinant Bacteriophage 11 and 12 Endolysins on Whole Cells and Biofilms of Staphylococcus aureus.

3/22/13

3/25/13

3/27/13

3/29/13

April

4/1/13

4/4/13

4/5/13

4/8/13

4/10/13

4/12/13

4/15/13

May

5/1/13

5/3/13

5/6/13

5/8/13

5/10/13

5/13/13

5/15/13

KK Yesterday's transformation of pIG13+CRO insert into E.Coli yielded several colonies on both the plate inoculated with 50 microL and the plate inoculated 100 microL. We plated 8 separate single colonies and will wait until Friday, when we will plan to PCR verify which of the colonies has our vector and insert.

KP 5/15/13 Today I re-streaked our ligation products. I re-streaked the control (pLat vector only) and the test (pLat vector and cro insert.) The hope, is that we can get individual colonies from the plates, so that we can sequence our plasmid to see if the vector took up the insert and if we have what we think that we do. We we will need primers IG57 and IG58 for our sequencing of this plasmid on Friday.

Cholera - Detection

From 2013.igem.org

Contents

March

3/15/13

3/18/13

3/20/13

3/22/13

3/25/13

3/27/13

3/29/13

April

4/1/13

4/4/13

4/5/13

4/8/13

4/10/13

4/12/13

4/15/13

May

5/1/13

5/3/13

5/6/13

5/8/13

5/10/13

5/13/13

5/15/13

5/17/13

5/20/13

5/22/13

5/24/13

5/27/13

5/29/13

5/31/13

June

6/3/13

6/5/13

6/7/13

6/10/13

6/12/13

6/14/13

6/17/13

6/19/13

6/21/13

6/24/13

6/26/13

6/28/13

July

7/1/13

7/3/13

7/5/13

7/8/13

7/10/13

7/12/13

7/15/13

7/17/13

7/19/13

7/22/13

7/24/13

7/26/13

7/29/13

7/31/13

August

8/2/13

8/5/13

8/7/13

8/9/13

8/12/13

8/14/13

8/16/13

8/19/13

8/21/13

8/23/13

8/26/13

8/28/13

8/30/13

September

9/2/13

9/4/13

9/6/13