PHB production

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

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Fig1,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]).