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Team:Tokyo Tech/Projects/rose pattern/index.htm
From 2012.igem.org
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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. 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. 9/4
Achievement
TEXT
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. 9/4
Project Overview
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 9/4
Production of PHB main culture
Protocol
1 Wash Erlenmeyer flasks with mild detergent then rinse with distilled water.
2 Measure 4% LB medium and add it to each Erlenmeyer flask inside clean bench.
3 Add 96% distilled water to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.
4 Set all flasks into autoclave
5 Clear the clean bench
6 Add 1/1000 Chloramphenicol and 4% Glucose solution (50%) after the medium is completely cooled.
7 Add 2% preculture solution into each flasks and shaking culture at 37 ° C for 72 hours.
Collection of PHBs in JM109
1 Weigh empty 50ml falcon tube without lid and make a record.
2 Add some culture solution into each tube.
3 Set the tubes into centrifuge and make sure that the label faces outside.
4 4 ° C ,5000G ,10min in centrifuge.
5 Remove the supernatant with electric pipettor then add culture solution and set in centrifuge again.
6 After adding all the culture solution and setting in centrifuge, remove the supernatant and add water, set in centrifuge again.
7 Remove the supernatant and add a little amount of water.
8 Cover the tubes with double layers of parafilms and fully freeze them.
Freeze drying
1 Poke several holes on the tubes’ parafilm with toothpick.
2 Set the tubes on the freeze drying machine.
3 Freeze dry for 3 days.
date9/4
| Disease | Target Range | Binding Site Location | Bottom Finger | Top Finger | Bottom AA (F3 to F1) | Top AA (F3 to F1) |
| Colorblindness | chrX:153,403,001-153,407,000 | 3627 | GTG GGA TGG | GAA GGG ACC | RNTALQH.QSAHLKR.####### | QDGNLGR.RREHLVR.####### |
| Familial Hypercholesterolemia | chr19:11,175,000-11,195,000 | 14001 | GGC TGA GAC | GGA GTC CTG | ESGHLKR.QREHLTT.####### | QTTHLSR.DHSSLKR.####### |
| Myc-gene Cancer | chr8:128,938,529-128,941,440 | 198 | GGT GCA GGG | GGC TGA CTC | VDHHLRR.QSTTLKR.RRAHLQN | ESGHLKR.QREHLTT.####### |
| Myc-gene Cancer | chr8:128,938,529-128,941,440 | 981 | GGA GAG GGT | GGC TGG AAA | QANHLSR.RQDNLGR.TRQKLET | EKSHLTR.RREHLTI.####### |
