"

From 2012.igem.org

Characterization & Experimental Design

Meeting 7/2/12

List of What Needs to get Done this Week:

• Research protocols on setting up and running experiments using biofilms
• Research flow and static protocols for biofilms
• Research ways to characterize silica matrix and acquire quantifiable data
• Create timelines for each assay researched
• Order reagents and get training on any equipment needed for assays

7/3/12

Researched stages of biofilm formation:

1. Initial Attachment - attachment occurs via van der waals forces

2. Irreversible Attachment - this is where the EPS forms

3. Maturation I - matrix formed with adhesion proteins

4. Maturation II - the full biofilm forms

5. Dispersion - bacteria released

Also researched shear stress modeling, chassis development, microsensors, and confocal microscopy assays.

Furthermore, research was done on different ways to characterize the silica coat. Three extremely helpful articles are listed below.

• Cell-Mediated Deposition of Porous Silica on Bacterial Biofilms
• Sol-gel synthsis and structure of silica hybrid materials
• Thin-film silica sol-gel coatings for neural microelectrodes

A powerpoint highlighting possible characterization methods is given here

After discussion with the team and our faculty advisor, Dr. Rickus, we have decided to use the scanning eletron microscopy (SEM), x-ray diffraction (XRD), and a live/dead assay (specifically, the Baclight Bacterial Viability Kit).

7/5/12

Researched possible competitors in the field of bioreactor filtration.

• Hydro Engineering - produces "Hydrokleen", which is a bioreactor filtration system that would generally be used in industrial plants and acts as a water recycler.
• Seven Trent Services - produces Tetra Amphidrone fixed sequencing Batch Biological Filter, a waste water treatment process that uses a biological filter to remove various pollutants
• Kinetics - this company makes a range of water treatment processes and machines that can be used in industry as well as consumer markets
• ABEC - produces bioreactors, but could not find more detail into what kind

7/8/12

Created a cell culture to have competent cells for a biofilm culture.

Also did research into developing a static biofilm assay to test how well our bacteria makes biofilm before and after our transformation. "Growing and Analyzing Static Biofilms" by Judisth H. Mernitt, Daniel E. Kadouri, and George A. O'Toole was helpful in determining how we would do this assay.

7/9/12

Performed the Growth Rate Assay on NEB Beta and DH5 Alpha E.coli strains to determine the time frame we will use to grow our static biofilms.A typical bacterial growth curve is shown below. This is what we expect our curves to look like.















Below is the results of the growth rate assay for NEB Beta and DH5 alpha. Overall, the plots show the expected shape with a lag, log, and stationary phase.














Also worked out the lab plan for the rest of the week.

7/10/12

We did the same Growth Rate Assay as yesterday, but with the XL-10 Gold E.coli strain because we think it will work better for the biofilm purposes.

Below is the results of the growth rate assay for XL-10 Gold. The plots show the expected shape with a lag, log, and stationary phase.

Here is the excel file containing all of the data from the three growth rate assays.
Also researched and developed the static biofilm assay that we can do to determine the biofilm rates of our non-transformed E.coli.

7/13/12

Performed the Static Biofilm Assay.

Had a team meeting, discussed goals for the following week:

• Write out protocols and email them to the entire team for revisions
• Plan a meeting to go through and troubleshoot the protocols together
• Find a way to determine the optimized concentration of curli
• Purchase all materials needed for future protocols

7/16/12

Researched how Membrane Aerated Bioreactor work and how we will use one for testing.
There are multiple types of Membrane Aerated Biofilms: MABR (the type we will be using in our experiments), Membrane biofilter, and extractive membrane bioreactor (EMB). These reactors can be in shell and tube or plate and frame configurations. We will be using the shell and tube configuration. The two limiting substrates are introduced at opposite ends of the bioreactor. The limiting substrates are C-substrate (carbon substrate) and/or oxygen. The biofilm thickness must be less than the oxygen penetration (50-200 micrometers) but stay less than 150 micrometers for best performance.

7/23/12

There were some modifications to the Static Biofilm Assay Protocol. Once the plates had grown for a long enough time period (every hour for 1-8 hours and then 24 hours and 48 hours) they were transferred to the 4 degree freezer. The plates were then removed, the excess liquid was dumped out, and the plates were then sprayed off with water and dumped out again. The plates were than stained with crystal violet with the following steps:

• add 125 microliters of crystal violet to each well plate
• let sit for 15 minutes
• wash off (Note: at this point the plates are stable for several weeks)

Analyzing Plates:

• 500 microliters of 80% ethanol | 20% acetone were added to each well
• 300 microliters (or enough until the well were full) were taken from the 24 well plate to the 96 well plates
• this was then analyzed using the spectrophotometer to determine the OD of each sample

7/26/12

Purpose: To make a solution to grow XL-10 gold strain of E.coli so that it can be integrated into a membrane aerated biofilm reactor (MABR) Materials:

• SOB - 250 mL
• 10 mM MgCl2 (.238 g)
• 20 mM glucose (.900 g)
• 250 microliters of cultured XL-10 gold culture

Procedure: Add all the ingredients together in a flask and swirl. Place in 37 degree room and shake at 220 rpm. Check OD.
Feeding Solution for current biofilm:

• 200 mL nutrient (glucose and nitrogen)
• 1mL solution 2 and 3
• 100 microliters solution 1