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• iGEM 2013

Contents 1 March 1.1 3/15/13 1.2 3/18/13 1.3 3/20/13 1.4 3/22/13 1.5 3/25/13 1.6 3/27/13 1.7 3/29/13 2 April 2.1 4/1/13 2.2 4/4/13 2.3 4/5/13 2.4 4/8/13 2.5 4/10/13 2.6 4/12/13 2.7 4/15/13 3 May 3.1 5/1/13 3.2 5/3/13 3.3 5/6/13 3.4 5/8/13 3.5 5/10/13 3.6 5/13/13 3.7 5/15/13 3.8 5/17/13 3.9 5/20/13 3.10 5/22/13 3.11 5/24/13 3.12 5/27/13 3.13 5/29/13 3.14 5/31/13 4 June 4.1 6/3/13 4.2 6/5/13 4.3 6/7/13 4.4 6/10/13 4.5 6/12/13 4.6 6/14/13 4.7 6/17/13 4.8 6/19/13 4.9 6/21/13 4.10 6/24/13 4.11 6/26/13 4.12 6/28/13 5 July 5.1 7/1/13 5.2 7/3/13 5.3 7/5/13 5.4 7/8/13 5.5 7/10/13 5.6 7/12/13 5.7 7/15/13 5.8 7/17/13 5.9 7/19/13 5.10 7/22/13 5.11 7/24/13 5.12 7/26/13 5.13 7/29/13 5.14 7/31/13 6 August 6.1 8/2/13 6.2 8/5/13 6.3 8/7/13 6.4 8/9/13 6.5 8/12/13 6.6 8/14/13 6.7 8/16/13 6.8 8/19/13 6.9 8/21/13 6.10 8/23/13 6.11 8/26/13 6.12 8/28/13 6.13 8/30/13 7 September 7.1 9/2/13 7.2 9/4/13 7.3 9/6/13 7.4 9/9/13 7.5 9/11/13 7.6 9/13/13 7.7 9/16/13 7.8 9/18/13 7.9 9/20/13 7.10 9/23/13 7.11 9/25/13 7.12 9/27/13 7.13 9/30/13

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

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

Yesterday's transformation of pIG13+CRO insert into E.Coli yielded

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.

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

9/9/13

9/11/13

9/13/13

9/16/13

9/18/13

9/20/13

9/23/13

9/25/13

9/27/13

9/30/13