Bronze. 

Our team has satisfied the following departments: 
Registered for iGEM, had a great summer, and attended the Giant Jamboree.
⎫ Completed the Judging Form.
⎫ Created and shared a Description of the team's project using the iGEM wiki, and documented the team's parts using the Registry of Standard Biological Parts. ⎫ Will have presented a poster and a talk at the iGEM Jamboree.
⎫ Created a page on our team wiki with clear attribution of each aspect of our project. This page clearly attributes work done by the members of our team and distinguishes it from work done by others.
⎫ Documented at least one new standard BioBrick Part or Device central to our project and submitted it to the iGEM Registry. We have also documented a new application of a BioBrick part from the 2014 iGEM year, which was the pBAD promoter plus strong RBS. Silver. In addition to the Bronze Medal requirements, our team has also satisfied these 3 following departments: ⎫ Experimentally validated that our new BioBrick Part or Device of our own design and construction works as expected. Yersinia pestis tolC, Pseudomonas tolC, and Proteus tolC are the new BioBrick parts that we designed. We documented the characterization of each part in the Main Page section of the Registry entry for that Part/Device. ⎫ Submitted these new parts to the iGEM Parts Registry. This part was different from the part we documented in Bronze medal criterion #6. ⎫ iGEM projects involved important questions beyond the bench, for example relating to ethics, sustainability, social justice, safety, security, and intellectual property rights. We referred to these activities as Human Practices in iGEM. Demonstrate how your team has identified, investigated and addressed one or more of these issues in the context of your project. An important issue that the WLC-Milwaukee team has addressed in this year’s project are the ethical questions surrounding the use of genetically modified organisms (GMOs). Many people within our faith consider genetic manipulation to be within man’s grasp, yet perhaps we haven’t fully understood the implications of such abilities. In an attempt to better understand current perception of the ethicality of GMOs in our faith, the Wisconsin Lutheran College - Milwaukee team surveyed both teachers and students of Wisconsin Evangelical Lutheran high schools. The findings of these surveys were essential to both investigating and addressing this issue are found HERE. Both students and faculty at the high schools were closer to strongly disagreeing with the statement: “The idea of genetic manipulation is unethical” than they were to agreeing with it. In order to educate both the faculty, and by extension the students of those in the Wisconsin Evangelical Lutheran synod, the WLC-Milwaukee team has put together a Biotechnology Curriculum. It poses topics for discussion with both teachers and faculty and asks how work such as genetic modification coincides with our faith. The curriculum also suggests that concepts such as genetic engineering and synthetic biology be explored to attain a better understanding of the scientific methods in light of our faith. Gold. In addition to the Bronze and Silver Medal requirements, our team also achieved these two following goals:  ⎫ Expanded on our silver medal Human Practices activity by demonstrating how we have integrated the investigated issues into the design and executed into our project. We also demonstrated an innovative Human Practices activity that related to our project (this typically involves educational, public engagement, and/or public perception activities; see the Human Practices Hub for information and examples of innovative activities from previous teams). Our team investigation into the ethicality of genetic modification was integrated into our project in two ways. Over the summer the team hosted the 2015 WLC Bioengineering summer camp, a week long camp designed to educate high school students about Biotechnology. Throughout the week activities such as lab protocols and lectures from campus professors were the focus of those attending. In the afternoon students had time to work on a project with their groups which focused on creating to presentation of a past iGEM project to present to the rest of the students on campus. At the end of the week, both instructors and the students in attendance deemed the camp a success. While the Bioengineering camp was aimed at high school students, the Wisconsin Lutheran College – Milwaukee team also wanted to create a means of education for students currently attending WLC. To meet this need this year’s team introduced Biotechnology Information Night. The evening consisted of presentations from a few of the iGEM seniors. A total of 17 students were in attendance. Topics such as iGEM, GMOs, and other applications of biotechnology were presented and the evening ended with discussion concerning the topics covered. Most importantly there was also discussion concerning the ethicality of genetic engineering and what it means to our faith. Many students participated in the latter part of this discussion and a few of the senior iGEM members had the opportunity to share their thoughts as well as the conclusions they had come to over their years of study and participating in iGEM. It was a great discussion and we look forward to hosting these again in the future!

Western Blots

In order to look for phages for specific organism’s TolC proteins, we had to first make sure the proteins were being produced. In all, we isolated the tolC genes for Salmonella, Vibrio cholera, Yersenia pestis, Proteus, Pseudomonas, Klebsiella, and E. coli K12. These were put under the control of the pBAD promoter and a strong E. coli RBS, and the E. coli tolC signaling sequence was put upstream the different organisms’ tolC genes. Using the Western Blots, we were able to show that the proteins were being transcribed, and transcription levels were raised in the presence of the pBAD promoter’s inducer- arabinose (at 0.1% working solution). Note: basal levels of transcription in Luria Broth are sufficient for function (see Kirby-Bauer assays which were done without the inducer) and phage binding (see the example phage plate in Modeling > Experimental Verification, which was also done without any inducer).

Kirby-Bauer Assays

The Kirby-Bauer assays were the second part of our expression tests. While the western blots tested for the production of the proteins, the Kirby-Bauer assay is testing whether or not the expressed TolC is having an impact on sensitivity to two efflux-resisted antibiotics: Novobiocin and Erythromycin. The Kirby-Bauer assay is a simple test that involves plating what would become a lawn of bacteria (using Top Agar); on top of this filter disks containing an antibiotic are placed. The antibiotics diffuse from the disks and inhibit the growth of bacteria within a certain distance based on the concentration of antibiotic at that distance and the minimum inhibitory concentration (MIC) of the antibiotic against the specific bacteria plated.

In the Kirby-Bauer assays, wild type (WT) E. coli, ΔtolC E. coli, and a ΔtolC E. coli with the pUC57 plasmid containing the pBAD promoter and the strong RBS (everything but a gene) were used as controls. The WT E. coli had smaller zones of clearing, indicating reduced sensitivity to the antibiotics compared to the two other controls, as expected. The rest of the samples were ΔtolC E. coli expressing a transgenic tolC gene contained on a pUC57 plasmid behind the same pBAD promoter-strong RBS combination. A Kirby-bauer showing zones of clearing similar in size to the WT E. coli was interpreted as TolC proteins being successfully assembled, inserted into the membrane, and successfully interfacing with other membrane proteins to form a functional trans-periplasmic efflux pump. Zones of clearing similar to the ΔtolC control were interpreted as a failure interface with other membrane proteins to form a fully functional trans-periplasmic efflux pump. It is impossible to say based on the Kirby-Bauer assays if these failures were due to failure to polymerize, insert into the membrane, or interacting with the other membrane proteins in the efflux pump.

Kirby-Bauer assays were analyzed by taking a picture of the plates from the same height (using a stand) and keeping a ruler in the image. The pictures were analyzed using the ImageJ software package. A conversion was set between pixels and centimeters using the “Set Scale” function. A circle was made to reflect the zone of clearing and its properties were exported as a .csv. Excel was used to convert from the circumference to the diameter of the zone of clearing, and each diameter was expressed as a percentage of the ΔtolC E. coli’s zone of clearing’s diameter.

Phage Solution Enhancement

Our project aims to act as a chassis for collecting and isolating new bacteriophages specific to the TolC proteins of pathogenic strains of bacteria, using a safe lab-strain of E. coli. To be used in such a way, we modified a preexisting protocol for isolating bacteriophages from the environment. In doing so, we added three rounds of what we referred to as “phage solution enhancement.” The idea behind this is that the environmental phage source is incubated with a larger volume of overnight-grown ΔtolC E. coli for ~20 minutes; this should be enough time for any bacteriophages capable of infecting these knockout-tolC cells to have an opportunity to bind a host bacterium, but not enough time to replicate and release new phage particles into the environment. These incubations are then chilled and centrifuged. The pellet created at the bottom contains the ΔtolC E. coli and any non-TolC specific bacteriophages which bound/infected the ΔtolC E. coli. The supernatant, which contains any bacteriophages which did not bind, is moved into a new tube, treated with chloroform to kill any remaining bacteria, and moved into another new tube. This becomes the new phage solution and the process is repeated twice more. The protocols are included in the table below.

In order to justify these steps, we attempt to quantify the effect of our process of phage solution enhancement. In order to test the protocol’s effectiveness, our experiment made use of two phages. One TolC-binding phage, TLS phage, and one non-TolC-binding phage we isolated ourselves called Phage #4 (we found that mipA, fhuA, fecA, and flgH knocokout E. colis, were insensitive to this phage). To begin the experiment we infected bacterial cultures (separately) in order to proliferate our phages; 1 mL of overnight grown WT E. coli (wild type) was mixed with 10 mL of LB and 250 µL of isolated phage solution. After incubating overnight at 37°C, these tubes were centrifuged. The supernatants were moved to new tubes; these were treated with chloroform, centrifuged, and the supernatants were again moved to new tubes (these are our starting phage solutions). From there four new tubes were labeled (TLS+, TLS-, 4+, and 4-). The “+” tubes became the “enhanced” tubes, and the “-“ tubes were left “unenhanced”; both “TLS” tubes were started with 5 mL of the TLS starting phage solution, and both “4” tubes were started with 5 mL of the Phage #4 starting phage solution. The “+” tubes were enhanced (see steps 2-9 under “TolC Specific Bacteriophage Screen” above) adding 30 mL of volume to the original 5mL of phage solution. To compensate for the gained volume, the “-“ tubes had 30 mL of sterile LB added.

Expecting high phage concentrations in some of our new phage solutions, we performed 1:10 serial dilutions (of which 100µL of the total 1000µL of each dilution would be used) on all of our new phage solutions down to a concentration of 10^-12. All these dilutions were mixed with 250 mL of overnight grown WT E. coli culture and 4 mL of molten LB top agar; the mixtures were immediately plated. After ~24 hours of incubation at 37°C, the plates were analyzed. Plates with a countable number of plaques (between 30 and 300) were chosen for each type of phage solution. These were used to calculate the number of plaque forming units per milliliter (PFU/mL). The “+” solutions’ PFU/mL’s were divided by those from the matching “-“ solution, to obtain a fractional change in phage concentration from the enhancement process. We expected a fractional change of approximately 1 from TLS+/TLS- and a number significantly less than 1 for Phage #4. Results are below. These results demonstrate the efficacy of the phage enhancement process.