Materials & Methods

Construction of pha-C1-A-B1 in Biobrick format

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fig3,construction of phaC1-A-B1

To construct a part that meets Biobrick format, we have modified the phaC1-A-B1 operon not to contain forbidden restriction enzyme sites. First, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K934001 BBa_K934001]). Fig3:construction of phaC1-A-B1

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Production trial of PHAs by past teams

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

iGEM10_Caltech

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

iGEM10_INSA-Lyon

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

iGEM08_Utah State

iGEM08_Hawaii

iGEM08_Virginia

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Protocol

A .PHB production on colonies and preparation before confirmation with Nile red under UV

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1 Preparation of LB agar medium plate containing Nile red and Glucose

2 Transformation of E.coli strain JM109 with pSB1C3 plasmid containing phaC1-A-B1 into strain JM109

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B.PHB production in cells and preparation before the confirmation with Nile blue A

[Back to "4-2 Confirmation of PHB accumulated in cells"]

1 Production of PHB

1.1 Acquire one colony of the transformed strains (JM109) with a platinum loop

1.2 Culture the colony in LB solution for 16hrs at 37 ° C

1.3 Measure LB medium (final 2.5%) and add it to each Erlenmeyer flask inside clean bench.

1.4 Add distilled water(final 95%) to each Erlenmeyer flask and cover the flasks with four-folded aluminum foil.

1.5 Set all flasks into autoclave

1.6 Add Chloramphenicol(final 25ug/ml) and glucose solution (50%) (final 20g/L) after the medium is completely cooled.

1.7 Add the solution of cultured cells into each flasks and shaking culture with air permeable lids at 37 ° C for 96 hours.

Fig3-2-2. air permeable lids

2 Preparation before the confirmation (with Nile blue A) under fluorescent microscope