Difference between revisions of "Part:BBa K1725332"

Latest revision as of 20:02, 19 September 2015

Member of the RBS library - derived from BBa_B0032

Parts K1725301-K1725332 are members of ribosome binding site library derived from Anderson-family RBS BBa_B0032. All 32 sequences were obtained by randomised PCR.

Design

For the construction of the RBS library, a master sequence based on the RBS B0032 was used. 4 nucleotides within the actual ribosome binding site were randomised giving 32 different B0032-derived RBS variants. The predicted efficiency of each RBS library member was estimated using RBS Library Calculator. Originally, RBS Library was designed for every gene in luxABGCDE operon (BBa_K1725352) in order to optimise bioluminescence in E. coli. 32 different BioBrick RBS variants were incorporated upstream of each of the lux genes by PCR, and different libraries were then combined together by BioBrick assembly. This method could potentially produce over 1 billion different variants of the lux operon. The developed method was relatively quick, and could easily be used to optimize other metabolic pathways where a suitable screen or selection for optimal performance is available.

Different variants of the lux operon in E. coli

Design of the RBS library for the lux operon

B0032-derived RBS library members

Estimated strengths of RBS library members for luxA gene; from left to right: K1725332 - K1725301

Randomised sequence locations are shown in red. Estimated strength corresponds to the Translation Initiation Rate values predicted by RBS library calculatorfor luxA gene.

Obtaining parts

RBS library parts are not submitted to the registry. For obtaining physical DNA of a particular RBS sequence or of the whole RBS library we offer de novo synthesis by PCR.
Primer sequence for obtaining RBS library for a specific gene:
aaataaGAATTCGCGGCCGCTTCTAGAGTCACACANRARRGTACTAGATG
5' extension to allow efficient EcoRI cleavage--biobrick prefix--RBS degenerate sequence--RBS scar--start codon
After the ATG, oligo should contain 18-24 nucleotides, complementary to the open reading frame being amplified. PCR is then performed as described in the [http://2015.igem.org/Team:Glasgow/Project/Overview/Protocols Protocols].

Sequence and Features