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

Recently, polyhydroxyalkanoates (PHA) have received a lot of attention for their potential use as bioplastic. They are naturally occurring biopolymers – under low nitrogen, phosphorus, or oxygen conditions, many bacteria produce protein- and lipid-coated granules of PHA for energy storage, and these granules can then in turn be harvested in large quantities as bioplastics. Bioplastics not only help reduce the reliance on petroleum-based plastics, but also have a wide range of applications in the agricultural and medical fields because they are biodegradable and biocompatible. The most common PHA is poly(3-hydroxybutyrate), or poly(3HB), which is produced by the bacteria Cupriavidus necator. Three genes – phaA, phaB, and phaC – make up the phaCAB operon that synthesizes the enzymes required for formation of poly(3HB), which is not elastic enough for general plastic applications.

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

3. Make sure the product under microscope