ELLA

• Content - some data, some of the discussions/outreach we had with people.

Outreach at South by Southwest (SXSW)

Jordan discussing our project with Matt Bolick, co-owner of Flat Track Coffee

Ella collecting coffee sample from Austin Java

In the spring of 2014, [http://www.kareningram.com Karen Ingram], invited the UT Austin iGEM team to create a presentation for the [http://sxsw.com/exhibitions/sxsw-create/participants-2014 South By Southwest (SXSW) 2014 Create event]. The SXSW festival is an annually held set of music, film and interactive events in Austin. The Create event was an interactive event that showcases up and coming maker/hacker/DIY culture. There were many relatively new team members, so the we saw this as an opportunity to not only teach the new members essential research techniques, but also to reach out to the community and raise awareness for the emerging field of synthetic biology. We decided to revive an old iGEM project, the "Caffeinated coli," and thought it would be interesting to measure the caffeine content of various different local house coffees.

Caffeinated Coli

[http://www.google.com/maps/d/viewer?mid=zlUDhks6KHxI.ketDvlvg6zis Click here for an interactive map of coffee shops we visited!]

Previously, the UT Austin iGEM team created and used the caffeinated coli to measure the caffeine content in beverages such as soda and energy drinks. The results showed that these engineered E. coli were very accurate at detecting the levels of caffeine in these formula-based drinks. We wondered how well they would do with something different, something that the vast majority of people in the city of Austin drink on a regular basis, coffee.

The [http://www.ibuyaustin.com Austin Independent Business Alliances'] slogan of [http://www.keepaustinweird.com "Keep Austin Weird,"] is a widely shared sentiment of Austinites, and emphasizes the need to promote local businesses. Thus, we thought it would be a great community outreach project to work with local Austin coffee shops, and do our part to "Keep Austin Weird!" Jordan, a senior member of the group, took charge and assembled a map of about 40 local coffee shops in Austin. The team split up one weekend to visit dozens of coffee shops, collecting samples of their house coffee. We were met with much enthusiasm by the coffee shop owners and employees, whose interest had been piqued when we described our experiment, often resulting in discussions of science, synthetic biology, and coffee.

The beginning of something great! Special thanks to Razan Alnahhas (second row, right) for mentoring and to Mike Hammerling (not pictured) and Ashley Kessel (second row, left) for assistance in sample collection.

Back at the lab, the team members got to work. Senior members divided their attention to mentor the new members in the techniques necessary to start cultures, work with the lab equipment, and other general laboratory safety.

Measuring Caffeine Content

Cultures of knockout strain grown with collected coffee samples.

Standard growth curve of knockout strain grown with different amounts of caffeine.

The E. coli we used are an engineered strain, previously made by the 2012 UT Austin iGEM team. This strain has had its normal guanine synthesis pathway knocked out, but contains a plasmid with a set of genes that enables the organism to synthesize guanine from xanthine and many of its derivatives, including caffeine. Without caffeine (or other xanthines), the strain cannot grow. When caffeine is added, the strain can demethylate the caffeine moleculesm yielding xanthine, and from there the E. coli can synthesize guanine. It then grows normally until the caffeine is completely metabolized, at which point the cells can no longer synthesize guanine, and cease to grow. Thus, by looking at the relative growth of the strain with different samples of coffee and comparing it to a standard curve of growth in solutions with known amounts of caffeine, we could reliably measure the amount of caffeine in the coffee.

The protocol was simple: We provided our Caffeinated coli with a diluted sample of coffee we had acquired from the various shops, and then compared the relative growth of each culture by measuring the absorbance at 600 nm (OD600 for short). The simplicity of the project was the key its success. It provided a glimpse into the exciting new world of synthetic biology for the new members and allowed them to learn vital skills that were used in day to day research during the summer. Our results can be seen in Figure 1, below.

Figure 1. Relative levels of caffeine in house coffee samples from around Austin. Data depict the amount of caffeine in the coffee relative to the average caffeine content for all samples tested, as determined using the caffeinated coli.

SXSW Create

In March 2014, we presented this data set at the SXSW festival. As part of this outreach, we explained our project and synthetic biology to a wide range of people. Many people who came by had little to no background in science or technology, and even most of the ones who did did not have a thorough understanding of what synthetic biology was or how it could be helpful. By discussing our experiments with bacteria and coffee, an interesting and easy-to-understand application, we were able to explain the basic concepts of synthetic biology and how it can be used in a beneficial way. Occasionally, these conversations would branch out into wider topics of science and technology.

People were particularly interested in the different types of coffee that yielded unexpected results or results that clashed with preconceptions. For example, we tested a Light Roast and Dark roast from the same coffee shop, Epoch. Most people, when asked if they knew the difference between the roasts would respond that the "dark/light roast had more caffeine", but in reality the caffeine content in the two roasts was indistinguishable; the light roast had a tiny bit more--but not enough to claim a significant difference.

--- Is this (below) an accurate description? If so, we should edit the tone a bit and we can include it:

Some people claimed that longer roasting destroyed the caffeine (and swore by cold-press coffee!), some people guessed that perhaps the dark roast sample had less coffee beans in it when it was brewed. Nevertheless, after much research by the team after the event, it was concluded that [http://en.wikipedia.org/wiki/Coffee_roasting coffee roasting] and [http://en.wikipedia.org/wiki/Coffee_preparation#Brewing coffee brewing] are largely variable processes, and caffeine content can vary significantly from batch to batch of beans. For more information about light roast vs. dark roast see this article at [http://www.scribblerscoffee.com/coffees_caffeine.htm Scribblers Coffee], which discusses the nuances between the two!

People also were extremely curious as to why the Wright Bros. Brew & Brew sample had a significantly higher amount of caffeine. This data point resulted in one of the owners of Wright Bros. Brew & Brew contacting us, to understand our process of determining caffeine content and why they were so far outside the mean. The owner, Matt Wright, concluded, after discussing our project and the coffee brewing process, that the specific pot of coffee was probably just not fully drained, i.e. there was less water in the batch than normal, resulting in a higher caffeine/volume content. This was just a starting point though as the conversation with Mr. Wright spanned the entire process of making coffee, quality control, and a little bit of the science behind the process. It also developed a nice contact point for future continuation of science and coffee.

However, similar conversations involving science, synthetic biology, coffee, and the community were had throughout the entire process. The various coffee shop owners and baristas were curious if they could employ science to help them make a better drink or a more customizable product. Some of the SXSW attendees were in the coffee-making business and were interested in thinking about whether such science could be used to understand where the caffeine goes during each step in the coffee-making process. And, a number of individuals who home brew coffee of kombucha or other organic drinks wondered if we could test their samples to determine the amount of caffeine they have in their beverages. We, of course, said yes!

Coffee bean counting contest

To involve everyone attending the event and raise more awareness for the iGEM team, we held a contest in which patrons attending the event could enter to win an E. coli plush upon following us on Twitter.

Special thanks to Karen Ingram for giving us this amazing opportunity to participate at SXSW Create 2014!

Photographs