The lac promoter, dxs and appY are from E. coli; LIMS1 is from lemon.
This part consists of a lac promoter (with lacZ' tag for blue-white selection) coupled to the limonene generation system consisting of dxs (deoxyxylulose phosphate synthase, rate controlling step of terpenoid biosynthesis in bacteria) + LIMS1 (limonene synthase 1 from lemon) + appY (a transcription factor known to increase terpenoid biosynthesis).
strong synthetic E. coli ribosome binding site with SacI site.
This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.
Note the presence of a SacI site overlapping the XbaI site, which is preserved when this biobrick is added to any other biobrick. At the time of writing, this biobrick is added as a short piece of DNA composed of two complementary oligonucleotides rather than being incorporated into a biobrick cloning vector by itself. It can be added upstream of a biobrick coding sequence, and its presence can easily be detected in miniprep DNA on a gel by using a SacI-SpeI or similar digest.
The DNA was amplified from E. coli BL21 genomic DNA using primers based on published sequence (Genbank accession J01636, gi:146575). The annotation shown here is based on that associated with this Genbank entry. The sequence shown here is derived by sequencing the construct.
This part (submitted in pSB1A2) consists of the lac promoter and lacZ' gene, encoding the N-terminal 76 amino acid residues of LacZ, sufficient to complement the lacZ-delta-M15 mutation for blue-white selection on Xgal plates. A SacI site has been introduced at the 3' end, overlapping the XbaI site of the Biobrick prefix. This is designed to be used as a cloning vector for making new biobricks. PCR primers can be designed with a SacI site in one primer and an SpeI site in the other. This removes the necessity for an excessively long non-complementary tail on one primer bearing either the full biobrick prefix or suffix. The PCR product can then be digested with SacI and SpeI for insertion into this plasmid, replacing the Plac-lacZ' cassette. Recombinant plasmids will then be white on IPTG/Xgal plates, whereas any that still contain the original insert will be blue. We have used this strategy to prepare several biobricks, including BBa_J33204, which contains the xylE gene encoding catechol-2,3-dioxygenase. (For making biobricks that contain lacZ', BBa_J33204 can be used in the same way; in this case, clones with plasmids that still contain xylE will turn yellow on addition of a drop of 10 mM catechol.)
Note that the SacI site overlaps the SpeI site. The Biobrick prefix ends ...TCTAGAG. When this is added to the CTC at the start of the sequence shown here, the SacI site, GAGCTC, is generated.
dxs coding sequence encoding 1-deoxyxylulose-5-phosphate synthase
This is the coding sequence of dxs from Escherichia coli JM109. It encodes 1-deoxyxylulose-5-phosphate synthase, thiamine-requiring, which catalyses teh first step in the biosynthesis of terpenoids. Overexpression of dxs has been reported to increase yields of carotenoids and other terpenoids [add reference here].
appY coding sequence encoding a DNA-binding transcriptional activator
This is the coding sequence of appY from Escherichia coli JM109. It encodes a transcriptional regulator related to anaerobic energy metabolism. Overexpression of the gene has been reported to result in enhanced lycopene production by E. coli (Kang et al., 2005).
Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases, J Lucker, et al, Eur. J. Biochem. 269: 3160???3171 (2002) Analysis of Lemon and Bergamot Essential Oils by HPLC with Microbore Columns, M Benincasa, et al, Chromatographia 30(5/6):271 (1990) Citrus fruit flavour and aroma biosynthesis, L Sharon-Asa, The Plant Journal, 36:664 (2003)
Limonene synthase 1 from Citrus limon (accession AF514287) Synthesised by Geneart. Limonene synthase converts farnesyl-diphosphate to + limonene, which is a component of lemon scent.
several codons were altered silently to remove several forbidden restriction sites. The N-terminal region (plastid targeting sequence) was removed, removing the first 51 amino acids and replacing codon 52 with the start codon ATG
Conversion of the iGEM parts registry to SBOL2.1