Team:HKUST-Hong Kong/overview111.html

1.3. Component details

1.3. Component details

Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000) oriR101 & repA101-ts is a set of low copy origin of replication derived from the pSC101 origin of replication. The repA101-ts gene codes for a heat-labile protein that is required in trans for the initiation of replication at oriR101. In our construct, our characterization has shown that plasmids with this origin of replication can only be maintained below than 300C, and partial maintenance of plasmid was observed within temperature range from 290C to 330C. This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by a nucleotide mutation.

split superfolder green fluroscent protein_split sfGFPsplit superfolder green fluroscent
protein_split sfGFP
sfGFP1-10 (BBa_K524001)
sfGFP11 ((BBa_K524002)

The sfGFPs are mutated variants of GFPs that has improved folding kinetics and resistance to chemical denaturants. Split sfGFPs at amino acid residues 214 and 215 have been reported to undergo spontaneous complementation to give green fluorescence. The two split constructs were produced from an existing biobrick – pBAD driven sfGFP BBa_I746908. CDS of sfGFP amino acid residues 1-214 were copied out for sfGFP1-10 using PCR and stop codon was added to the end. The sfGFP11 was produced in a similar fashion, with a start codon added to the front of the CDS of amino acid residues 215 to 238.

Essential gene nadE (BBa_K524003)
nadE is a vital gene in E. Coli. It codes for NAD+ synthetase. In principle, removal of such gene from the genome would cause addiction of bacteria to a plasmid that has a copy of the gene. CyaR (a sRNA) regulates the expression of nadE post-transcriptionally. This feature is retained in our construct. Transcription of nadE operon requires the sigma-70 factor and is terminated by downstream extragenic sites. The nadE gene was cloned out from the genome of strain BL21(DE3), and was completed the nadE by having B0015 terminator assembled to its end.

Replication initiator pi protein and ori-gamma from R6K plasmid
ori-gamma is one of three replication origins (the other two being alpha and beta) of the R6K origin. Initiation of replication at ori-gamma requires the pi protein in trans, which is encoded by the pir gene. Yet doubling the concentration of pi protein would effectively shut down the replication as well. Expression of pi protein is autogenously regulated. The pir construct was cloned out from the genome of strain BW25141 (courtesy of The Coli Genetic Stock Center) and standardized. The ori-gamma was adopted from the R6K origin of replication BBa_J61001.

iGEM 2010 Slovenia Split/FRET constructs
The split CFP and YFP from the biobricks of Slovenia team last year were used as alternative reporters. The idea is to put one of the terminal fragments of a split fluorescence protein into the pDummy, and swap out the essential nadE gene from the genome with the other terminal fragment. Driven by pLac R0010, both fragments should express simultaneously when induced by IPTG and fluorescence signal would be observed as an indicator of successful recombination.