Team:Tokyo Tech/Projects/PHAs/detail/index.htm

From 2012.igem.org

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{{tokyotechcss}}
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{{tokyotechmenubar}}
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<div id="tokyotech" style=" font:bold ;left ; font-size: 50px; color: #1E90FF; padding: 10px;">
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PHA production detail</div>
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</div class="whitebox">
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__NOTOC__
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<div class="whitebox">
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<div id="tokyotech" style=" font:bold ;left ; font-size: 15px; color: #000000; padding: 10px;">
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=Other Teams=
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=Production trial of PHAs by past teams=
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[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]
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We introduce some attempts in the past iGEM to show how great our work is in iGEM.
 +
 
[https://2010.igem.org/Team:Caltech iGEM10_Caltech]
[https://2010.igem.org/Team:Caltech iGEM10_Caltech]
{| class="wikitable" cellpadding="4"
{| class="wikitable" cellpadding="4"
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| align="center" style="background:#f0f0f0;"|'''experience'''
| align="center" style="background:#f0f0f0;"|'''experience'''
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K338003 BBa_K338003]||PHA Synthase Composite, Part 1/2||planning||- |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K338003 BBa_K338003]||PHA Synthase Composite, Part 1/2||Planning||
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K338004 BBa_K338004]||PHA Synthase Composite, Part 1/2||Available||None|
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K338004 BBa_K338004]||PHA Synthase Composite, Part 1/2||Available||None
|}
|}
They couldn’t prove that their engineered bacteria produced PHB according to the team wiki.  
They couldn’t prove that their engineered bacteria produced PHB according to the team wiki.  
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| align="center" style="background:#f0f0f0;"|'''experience'''
| align="center" style="background:#f0f0f0;"|'''experience'''
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K342001 BBa_K342001]||Pha C (poly-β-hydroxybutyrate polymerase)||available||None|
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K342001 BBa_K342001]||Pha C (poly-β-hydroxybutyrate polymerase)||Available||None
|}
|}
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They registered and submitted a part, which contains only phaC sequence, as worked. But the team wiki shows that the part doesn’t meet the standard which is required in iGEM because they couldn’t cut their part with Xba1.
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They registered and submitted a part, which contains only phaC sequence, as worked. But the team wiki shows that the part doesn’t meet the standard which is required in iGEM because they couldn’t cut their part with XbaI.
[https://2009.igem.org/Team:Duke iGEM09_Duke]
[https://2009.igem.org/Team:Duke iGEM09_Duke]
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| align="center" style="background:#f0f0f0;"|'''experience'''
| align="center" style="background:#f0f0f0;"|'''experience'''
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K282000 BBa_K282000]||phaAB||available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K282000 BBa_K282000]||phaAB||Available||None
|}
|}
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[https://2008.igem.org/Team:Utah State iGEM08_Utah State]
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[https://2008.igem.org/Team:Utah_State iGEM08_Utah State]
{| class="wikitable" cellpadding="4"
{| class="wikitable" cellpadding="4"
| align="center" style="background:#f0f0f0;"|'''Part number'''
| align="center" style="background:#f0f0f0;"|'''Part number'''
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| align="center" style="background:#f0f0f0;"|'''experience'''
| align="center" style="background:#f0f0f0;"|'''experience'''
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K089001 BBa_K089001]||phaA gene||planning|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K089001 BBa_K089001]||phaA gene||Planning||
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K089002 BBa_K089002]||phaB gene||planning|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K089002 BBa_K089002]||phaB gene||Planning||
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K089003 BBa_K089003]||phaC gene||planning|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K089003 BBa_K089003]||phaC gene||Planning||
|}
|}
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[https://2008.igem.org/Team:Hawaii iGEM08_Hawaii]
[https://2008.igem.org/Team:Hawaii iGEM08_Hawaii]
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| align="center" style="background:#f0f0f0;"|'''experience'''
| align="center" style="background:#f0f0f0;"|'''experience'''
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125801 BBa_K125801]||RBS-phaA||available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125801 BBa_K125801]||RBS-phaA||Available||None
|-
|-
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125802 BBa_K125802]
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125802 BBa_K125802]
||RBS-phaB||Planning|||
||RBS-phaB||Planning|||
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125803 BBa_K125803]||RBS-phaC||Available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125803 BBa_K125803]||RBS-phaC||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125804 BBa_K125804]||RBS-phaE||Available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125804 BBa_K125804]||RBS-phaE||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125501 BBa_K125501]||phaA BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)||Available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125501 BBa_K125501]||phaA BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125502 BBa_K125502]||phaB BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)||Available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125502 BBa_K125502]||phaB BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125503 BBa_K125503]||phaC BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)||Available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125503 BBa_K125503]||phaC BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125504 BBa_K125504]||phaE BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)||Available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K125504 BBa_K125504]||phaE BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)||Available||None
|}
|}
[https://2008.igem.org/Team:Virginia iGEM08_Virginia]
[https://2008.igem.org/Team:Virginia iGEM08_Virginia]
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| align="center" style="background:#f0f0f0;"|'''experience'''
| align="center" style="background:#f0f0f0;"|'''experience'''
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156031 BBa_K156031]||RBS + phaA + double terminator||available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156031 BBa_K156031]||RBS + phaA + double terminator||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156033 BBa_K156033]||RBS + phaB1 + double terminator||available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156033 BBa_K156033]||RBS + phaB1 + double terminator||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156034 BBa_K156034]||RBS + phaC1 + double terminator||available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156034 BBa_K156034]||RBS + phaC1 + double terminator||Available||None
|-
|-
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|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156012 BBa_K156012] ||phaA (acetyl-CoA acetyltransferase)||available|||
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|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156012 BBa_K156012] ||phaA (acetyl-CoA acetyltransferase)||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156013 BBa_K156013]||phaB1 (acetyacetyl-CoA reductase)||available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156013 BBa_K156013]||phaB1 (acetyacetyl-CoA reductase)||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156014 BBa_K156014]||phaC1 (Poly(3-hydroxybutyrate) polymerase)||available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156014 BBa_K156014]||phaC1 (Poly(3-hydroxybutyrate) polymerase)||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156021 BBa_K156021]||Promoter + RBS + phaA + double terminator||available|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156021 BBa_K156021]||Promoter + RBS + phaA + double terminator||Available||None
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156018 BBa_K156018]||Promoter + RBS + phaA||planning|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156018 BBa_K156018]||Promoter + RBS + phaA||Planning|| |
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156019 BBa_K156019]||Promoter + RBS + phaB1||planning|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156019 BBa_K156019]||Promoter + RBS + phaB1||Planning|| |
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156020 BBa_K156020]||Promoter + RBS + phaC1||planning|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156020 BBa_K156020]||Promoter + RBS + phaC1||Planning|| |
|-
|-
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156022 BBa_K156022]
| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156022 BBa_K156022]
-
||Promoter + RBS + phaB1 + double terminator||planning|| |
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||Promoter + RBS + phaB1 + double terminator||Planning|| |
|-
|-
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156023 BBa_K156023]||Promoter + RBS + phaC1 + double terminator||planning|| |
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| [http://partsregistry.org/wiki/index.php?title=Part:BBa_K156023 BBa_K156023]||Promoter + RBS + phaC1 + double terminator||Planning|| |
|}
|}
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=Production of PHAs by <I>E.coli</I>=
 
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(ⅰ)
 
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We observed the accumulation of PHB in the <I>E.coli</I> colonies on Nile red positive medium under UV .Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red produces a strong orange fluorescence (emission maximum,598nm) with an excitation wavelength of 543nm (maximum) upon binding to PHA-granules in cells of R.eutropha (Degelau et al.1995) H16. Nile red in the agar medium doesn’t affect the growth of the cells, and the occurrence of PHAs in the colonies can be directly monitored.
 
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This method is quite sensitive and results in fluorescent colonies of PHA-positive strains. So we cultured the transformant on LB agar medium plates with 0.5μg/ml Nile Red and 2% glucose at 37℃ for  30 hours, then we transferred the plates to 4℃ room. After  115 hours, colonies with PHB would be stained Red by Nile red when observed under UV. This showed that the transformant had stored PHB.FIG1 is the photograph of <I>E.coli</I>(with phaC1AB1 gene) colonies on Nile Red positive medium taken under UV. The fluorescent area in the figure showed the accumulated PHBs stained by Nile red in cells. This indicated that part BBa_K934001 functioned correctly.FIG2 is the photograph of negative control cells. In this figure we observed that there was no remarkable fluorescent area.
 
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(ⅱ)
 
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To confirm the accumulation condition of PHB in <I>E.coli</I> with a microscope, we stained the PHB with Nile blue A reagent. Nile blue A is also used to detect the existence of PHBs and has no toxicity to the cells. We carried out the observation after the procedures stated below. First，We cultured a small amount of the transformant (3ml) in LB medium for 17 hrs. Then，we added the cultured trandformant(1%) into LB medium(with 2% glucose) and shaking cultured it under aerobic condition with an air permeable lid for 96 hrs. After the cultivation we recovered the bacteria with PHA accumulation by centrifugation. We were able to get the dried cells after the procedure of lyophilization. Before taking the photographs，we stained the dried cells with 1% Nile blue A solution for 8 minutes and washed the extra Nile blue with 8% acetic acid solution afterwards. We then took photographs of the sample under fluorescence microscope. FIG1 is the photograph of <I>E.coli</I>(with phaC1AB1 gene) colonies on Nile Red positive medium taken under UV. The fluorescent area in the figure showed the accumulated PHBs stained by Nile red in cells. This indicated that part BBa_K934001 functioned correctly.FIG2 is the photograph of negative control cells. In this figure we observed that there was no remarkable fluorescent area.
 
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To confirm that PHB is stored in <I>E.coli</I>, we took photographs of the bacteria under microscope after the procedures stated below. First，We cultured a small amount of the transformant (3ml) in LB medium for 17 hrs. Then，we added the cultured trandformant(1%) into LB medium(with 2% glucose) and shaking cultured it under aerobic condition with an air permeable lid for 96 hrs. After the cultivation we recovered the bacteria with PHA accumulation by centrifugation. We were able to get the dried cells after the procedure of lyophilization. Before taking the photographs，we stained the dried cells with 1% Nile blue A solution for 8 minutes and washed the extra Nile blue with 8% acetic acid solution afterwards. We then took photographs of the sample under fluorescence microscope.
+
[[https://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#Construction_of_phaC1-A-B1_in_Biobrick_format Back to "Construction of phaC1-A-B1 in Biobrick format"]]

Latest revision as of 09:23, 22 October 2012

bar




Production trial of PHAs by past teams

[Back to "Construction of phaC1-A-B1 in Biobrick format"]

We introduce some attempts in the past iGEM to show how great our work is in iGEM.

iGEM10_Caltech

Part number Description States experience
BBa_K338003PHA Synthase Composite, Part 1/2Planning
BBa_K338004PHA Synthase Composite, Part 1/2AvailableNone

They couldn’t prove that their engineered bacteria produced PHB according to the team wiki.


iGEM10_INSA-Lyon

Part number Description States experience
BBa_K342001Pha C (poly-β-hydroxybutyrate polymerase)AvailableNone

They registered and submitted a part, which contains only phaC sequence, as worked. But the team wiki shows that the part doesn’t meet the standard which is required in iGEM because they couldn’t cut their part with XbaI.

iGEM09_Duke

Part number Description States experience
BBa_K282000phaABAvailableNone

iGEM08_Utah State

Part number Description States experience
BBa_K089001phaA genePlanning
BBa_K089002phaB genePlanning
BBa_K089003phaC genePlanning

iGEM08_Hawaii

Part number Description States experience
BBa_K125801RBS-phaAAvailableNone
BBa_K125802 RBS-phaBPlanning
BBa_K125803RBS-phaCAvailableNone
BBa_K125804RBS-phaEAvailableNone
BBa_K125501phaA BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)AvailableNone
BBa_K125502phaB BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)AvailableNone
BBa_K125503phaC BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)AvailableNone
BBa_K125504phaE BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)AvailableNone

iGEM08_Virginia

Part number Description States experience
BBa_K156031RBS + phaA + double terminatorAvailableNone
BBa_K156033RBS + phaB1 + double terminatorAvailableNone
BBa_K156034RBS + phaC1 + double terminatorAvailableNone
BBa_K156012 phaA (acetyl-CoA acetyltransferase)AvailableNone
BBa_K156013phaB1 (acetyacetyl-CoA reductase)AvailableNone
BBa_K156014phaC1 (Poly(3-hydroxybutyrate) polymerase)AvailableNone
BBa_K156021Promoter + RBS + phaA + double terminatorAvailableNone
BBa_K156018Promoter + RBS + phaAPlanning
BBa_K156019Promoter + RBS + phaB1Planning
BBa_K156020Promoter + RBS + phaC1Planning
BBa_K156022 Promoter + RBS + phaB1 + double terminatorPlanning
BBa_K156023Promoter + RBS + phaC1 + double terminatorPlanning


[Back to "Construction of phaC1-A-B1 in Biobrick format"]
Retrieved from "http://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm"