Story

PHA is a kind of bio-plastic produced by microbes.It's standard extract can be got after being dissolved in chloroform. In this project, we will reproduce the rose come out in the lines of the famous drama 'Romeo and Juliet' by the synthesis of PHA. PHA can be detected by Nile Red reagent by being dyed red, which looks exactly like rose.

Achievement

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Introduction

What's PHA

PHAs are the abbreviation of Polyhydroixyalkanoates, which are linear polyesters produced in nature by bacterial fermentation of sugar and lipids. They are produced by the bacteria to store carbon and energy. PHAs are also a kind of bio-plastics, which are more sustainable because they can be biodegraded in the environment a lot more faster than fossil-fuel plastics. PHBs are the abbreviation of polyhydrobutyrates ,which are a kind of polyhydroalkanoates. In our project we used the poly-3-hydroxybutyrate（P3HB）form of PHBs, which is the most common type of polyhydroxyalkanoate synthesized by the gene phaC1-A-B from Ralstonia eutropha H16.

As long as we know ,there was no team succeeded in synthesizing PHAs in iGEM format before. But this year we are very excited to tell you that we made it and we are going to show it to all of you.

synthetic mechanism

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Project Overview

Abastract

In our project, we will reproduce the rose come out in the lines of the famous drama Romeo and Juliet by the synthesis of PHAs. PHAs can be detected by Nile-Red reagent by stained red, which looks exactly like rose. If we can control the expression of the PHA by using reaction diffusion, the reproduciibility of the rose will reach a new level. And the area of PHAs stained red by Nile-Red reagent may even looks like a rose parterre. As E.coli is an unexpensive and easily available bacterium, it's commonly considered to be the ideal PHA producer. First of all, we must cut and paste the right gene sequences of PHA synthesis into E.coli .We take the gene phaC1-A-B from R.eutropha and paste it into the vector of IGEM format and then put this special plasmid into E.coli. Then we culture these E.coli on our LB medium with Nile Red. After that ,we will culture the colonies overnight to make sure that PHA can accumulate without disturbance.If things go well the Nile-Red reagent in the colony will turn red after detecting the existence of PHA.

Pre-experiment

Before the igem format vector coming，we actually synthesized PHA by using the vector PSB1C3 on E.coli JM109.We synthesized PHA by following steps. 1.Take the right gene phaC1-A-B from R.eutropha, and increase it by PCR 2.Insert the gene into the PSB1C3 vector by using a series of genetic operation including ligation and ethanol precipitation 3. Put the plasmid into E.coli JM109 by using the technique of transformation and plant it on LBmedium with Cm and Nile-Red then culture it overnight. 4. Check whether colonies are stained orange under UV 5. Produce PHAs by quantity by main culture the E.coli 6. Collect the strains and freeze dry it. 7. Stain the product and Measure the weight

Assay In order to check whether the product is PHA or not ,we used several ways of assay. 1. Read the gene sequence of the gene 2. See whether the product can be stained by Nile-Red or Nile-Blue