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Description Design Experiments Notebook Characterization Results Improve Modeling

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Parts Overview

SAFETY

HUMAN PRACTICES

Human Practices Education & Engagement

PROJECT DESIGN

PROBLEM & CONCEPT SELECTION

UV damage is a serious problem in many areas across the globe. From a young age, we are told to apply and reapply sunscreen, find shade, and monitor the amount of time spent in the sun. However, more than 2 people die of UV-induced skin cancer each hour in the US [1]. Our team decided to focus on this serious issue in today's society. Through our iGEM project, we hope to inform the public about the effects of UV on DNA by creating an educational kit that will be employable in primary and secondary school classrooms. To create such a kit, we will transform a plasmid with a promoter for a gene initiated during the early stages of SOS induction (uvrA) upstream of a green fluorescent protein (GFP) gene, which serves as a reporter molecule, into E. coli K12 strain. This system should fluoresce green when the DNA-damage repair mechanism is activated by UV exposure. Along with this visual demonstration, the kit will include a student worksheet and teacher's manual to emphasize the science taking place and real-life impact.

PLASMID DESIGN

Our biosensor involves an ampicillin-resistant plasmid with a native uvrA promoter and a sfGFP reporter gene. The purpose of this plasmid is to create a green visual output when UV-induced DNA damage occurs. The native uvrA promoter contains the sequence for the uvrA SOS box, a sequence where LexA, a repressor protein for the global SOS system in E. coli, binds to the uvrA promoter sequence. When UV induction occurs, LexA will be cleaved and free the uvrA promoter region for cellular machineries to express the downstream GFP gene. This plasmid is shown in the figure below.

Native uvrA - sfGFP

Figure 1: Plasmid map of uvrA.

We also designed two other plasmids to serve as controls for our main assay, which validated the function of our native uvrA - sfGFP plasmid. One of the controls, the uvrA promoter plasmid, contained only the native uvrA promoter and ribosome binding site (RBS) in an ampicillin-resistant backbone. This plasmid serves as a negative control for fluorescence since any fluorescence measured from this plasmid was a result of autofluorescence from our E. coli cells. The other control in this experiment was our constitutive GFP plasmid, which served as our positive control and had the sfGFP gene downstream of a constitutive pJ23119 promoter. This plasmid should not have been regulated by the SOS system and was expected to constitutively produce GFP. These plasmids are shown in the figures below.

Negative Control: Empty uvrA promoter

Figure 2: Plasmid map of uvrA promoter.

Positive Control: Constitutive promoter - sfGFP

Figure 3: Plasmid map of constitutive uvrA.

PRODUCT DESIGN

The educational kit contains the following materials:

• Glycerol stock of cells transformed with uvrA-GFP plasmid

• Glycerol stock of cells transformed with constitutive plasmid

• Petri dishes

• LB + Agar

• Ampicillin

• Serological pipette tips

We created a student guide with a list of materials, procedure, and student worksheet for the educational kit. These items are supposed to help learning about UV damage and exposure.

UV Exposure Experiment Student Guide

REFERENCES

[1] Skin Cancer Facts & Statistics. (2019). Retrieved from https://www.skincancer.org/skin-cancer-information/skin-cancer-facts/.