Synthetic biology is the foundation of numerous key fields such as medicine. However, the concepts of synthetic biology are not incorporated into the standardized K-12 curriculum. For educational outreach, we planned a Genetic Circuits Learning Module consisting of a ~30 minute presentation introducing the students to synthetic biology and the components of genetic circuits. The students would then complete a hands-on activity in which they design their own genetic circuit, created from predetermined components, then presented with a plate streaked with E. coli cells containing their designed plasmid. We determined that introducing students to genetic circuits was crucial to an effective immersion in synthetic biology because they serve as the building blocks of the vast, interdisciplinary applications of the field. We intend our lesson to be targeted at 9th grade/first year biology students.

Synthetic Biology Lecture

The goal of our lecture is to complement our genetic circuits activity and expose students to a concept that is likely not to be touched on in their general education that they may take interest in. Introducing students to the field of synthetic biology also encourages them to consider science in the context of something with tangible applications and even a career instead of the standard science curriculum which can seem very abstract. The lecture begins by introducing the students to our UOregon iGem team and what we have been working towards over the course of the year. We then move on to present a few of the applications and fields associated with synthetic biology. We then discuss crucial aspects of biology and their connection to genetic circuits, such as the function of DNA, as well as the four main components of a genetic circuit, the promoter, RBS, gene, and terminator. We continue by introducing plasmids and the procedure for assembling them and the method for cell incorporation of the genetic material. We conclude by introducing our genetic circuits activity.

Introduction to Synthetic Biology: Genetic Circuits Activity

The goal of our activity is to create a hands-on experience that parallels the wet lab experience of true synthetic biologists and encourage the students to practice their observation skills and utilization of microscopes, a fundamental piece of equipment in many labs. The students will choose paper circuit pieces with the nucleotide sequence on them. They have the choice between a strong promoter and rbs pair or a medium promoter and rbs pair, but the strength is kept blind. The students also choose one of three gene options which correspond to different colors of fluorescent genes. They are provided with only one terminator choice. There are six possible variations. The combinations were limited so that we could prepare plates for every possible choice. After taping together their circuit at the correct overhangs, the students are provided with the corresponding plate with E Coli cells expressing the plasmid to their sequence and make observations of the plate under the microscope. The goal is to determine the identity of their fluorescent gene and whether they chose the strong or weak promoter/rbs pair based on the color and brightness of fluorescence. They will be provided reference photos and a key to check their answers. This activity also allows the students to practice reading and comparing nucleotide sequences to identify arrangements and properties.

Wet Lab Experimentation

In preparation for the activity we designed genetic circuits from DNA segments supplied by the iGem Distribution Kit with matching overhangs and cuttable with BsaI. We initially decided on an orange, green, and blue fluorescent, TannenRFP, SYFP2, and mCerulean3 respectively as well as a strong and medium promoter from the constitutive promoter family and a strong and weak rbs, BBa_B0032_m0 and a weaker derivative. A separate terminator was not necessary because there is one built into the plasmid backbone, AE_lacZ pDest. We obtained cultures of each DNA part via electroporation into NEB 10-beta from the Distribution Kit. We attempted to assemble the target plasmids via golden gate assembly, however the procedure consistently failed. We explored the efficacy of each golden gate either via a gel or by starting a culture via heat shock into NEB Turbo. No fluorescence was ever observed. To trouble shoot, we tried increasing the length of the golden gate, we tried replacing the backbone and adding a terminator, and tried using chromoproteins instead of fluorescents; all were unsuccessful. We hypothesize that this failure is due to insufficient concentrations of the promoter and rbs in the golden gate because the target DNA sequences are contained within a much larger plasmid. This makes obtaining a relative concentration of DNA sequence needed for the golden gate extremely difficult. As such, we were not able to produce usable plates for our educational event, but we hope to continue making progress on this project within the next year.