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Contents 1 TITLE: The UV-responsive Wintergreen Odor Generator 2 ABSTRACT 2.1 INTRODUCTION 3 PARTS 3.1 Part A:BBa_J22106 rec A (SOS) Promoter 3.2 Part B:BBa_J45119 Wintergreen odor enzyme (BwMST1) generator 4 RESULTS 4.1 Growing Part A and Part B 4.2 Enzyme digestion of Part A and Part B 4.3 Enzyme digestion after ligation 5 STEP 5.1 STEP1: TRANSFORMATION AND GROWING 5.2 STEP2: MINIPREP 5.3 STEP3：RESTRICTION DIGEST 5.4 STEP4：LIGATION 5.5 STEP5：TRANSFORMATION 5.6 STEP6：MINIPREP 5.7 STEP7：RESTRICTION DIGEST 5.8 STEP8：DNA SEQUENCING 5.9 STEP9：UV INDUCTION 5.10 STEP10：DESGIN AND COMPLETE

TITLE: The UV-responsive Wintergreen Odor Generator

ABSTRACT

The UV-responsive wintergreen odor generator catalyzes the conversion of the precursor salicylic acid to the wintergreen odor methyl salicylate under extensive UV irradiation. The biosynthetic device is composed of two transcriptional devices: Rec A (SOS) promoter (BBa_J22106) and a wintergreen odor enzyme generator (BBa_J45119). The odor generator takes as input extensive UV irradiation and produce as output an enzyme that catalyzes production of an odor from a chemical precursor.

INTRODUCTION

Ultraviolet (UV) light is the spectrum consists of electromagnetic waves with frequencies higher than those that humans identify as the color violet. These frequencies are invisible to humans, but visible to a number of insects and birds.

UVC The shortest one of UV rays ，usually do not penetrate the Earth’s ozone layer.
UVB Longer waves that reach skin’s surface – can cause surface tanning burning ,and signs of aging.
UVA Even longer waves that can penetrate deep into the skin’s surface ,releasing free radicals and causing DNA changes that can result in skin cancers.

Recently, because the ozone layer has been destroying by some artificial chemical substances like CFC (Freon),the increase of UV irradiation appears on the earth. Extensive UV irradiation is harmful to human being, such as

• 1) Weaken the immune system of human
• 2) Increase the risk of skin cancer, cataracts
• 3) Make a heavy damage in genes

We designed a biological system in E.coli to detect extensive UV irradiation. If the toolkit is under extensive UV irradiation, it will produce a wintergreen odor.Then, This special odor will warn people of the UV irradiation. In order to increase the attraction of our product, we make E.coli culture medium with the different color using red, yellow and blue pigments.

PARTS

Part A:BBa_J22106 rec A (SOS) Promoter

Extensive UV irradiation impings on heavily DNA damage and results in a single-strand DNA occuring, which activates RecA protein.Then,RecA protein leads to the expression of about 20 different repair proteins to rescue DNA damage.RecA protein is the central regulator in SOS repair system.Rec A (SOS) Promoter is available in 2004 registry part plate.

Part B:BBa_J45119 Wintergreen odor enzyme (BwMST1) generator

BBa_J45119 takes as input a transcriptional signal (PoPS) and produce as output the BMST1 enzyme that catalyzes production of methyl salicylate from salicylic acid. Methyl salicylate has a wintergreen smell. Wintergreen odor enzyme (BMST1) generator is availabe in 2005 registry part plate.

RESULTS

Growing Part A and Part B

After Growing Part A

Enzyme digestion of Part A and Part B

Enzyme digestion after ligation

STEP

STEP1: TRANSFORMATION AND GROWING

(Make sure we have enough copies of BBa_J22106 and BBa_J45119.)

STEP2: MINIPREP

STEP3：RESTRICTION DIGEST

STEP4：LIGATION

STEP5：TRANSFORMATION

STEP6：MINIPREP

STEP7：RESTRICTION DIGEST

STEP8：DNA SEQUENCING

STEP9：UV INDUCTION

STEP10：DESGIN AND COMPLETE