dTomato, ModLac and Dioxygenase (inc RBS, NahR/Psal promoter system)
BBa_J61051, nahR with nahR and sal promoters (Pr and Psal), codes for the inducible repressor NahR, which suppresses Psal in the absence of salicylate. This biobrick makes it possible to regulate the expression of the red fluorescent protein, dTomato (BBa_K1688004), the modified laccase, ModLac (BBa_K1688006), and a dioxygenase.
dTomato and ModLac with HlyA tag (inc RBS, NahR/Psal promoter system)
BBa_J61051, nahR with nahR and sal promoters (Pr and Psal), codes for the inducible repressor NahR, which suppresses Psal in the absence of salicylate. This biobrick makes it possible to regulate the expression of the red fluorescent protein, dTomato, and the modified D439A/M510L CueO laccase (ModLac) with attached N-His6 tag and C-export tag (HlyA, BBa_K554002).
Catechol 1,2 Dioxygenase + HlyA export tag
Catechol 1,2 dioxygenase (BBa_K1092003) fused to HlyA export tag (BBa_K554002), but with an introduced frameshift due to improper assembly.
Primers for restriction-free cloning were designed in such a way as to remove the STOP codon of the catechol 1,2 dioxygenase and the scar left from 3A assembly. However, due to a design error, an AG insert between the two parts was introduced, resulting in a stop codon at the beginning of the HlyA sequence.
ModLac laccase with His-tag and HlyA export tag (inc RBS)
D439A/M510L CueO mutant sequence received from Professor Kunishige Kataoka and synthesized with His-tag and HlyA export tag.
A modified laccase (D439A/M510L CueO) with N-His6 tag attached to it via a linker sequence and an export tag (HlyA, BBa_K554002) attached to its C-terminus.
The modified CueO was synthesized with iGEM prefix and suffix, RBS(BBa_B0034) and a M-scar after the RBS in order to guarantee the function of the RBS. The gene is followed by a KpnI restriction site with glycines and serines on each side, which are small amino acids which increase the flexibility of the protein. This is needed for the export tag HlyA , to make it easier for the resulting signal protein to be folded properly. A second KpnI restriction site is put after the HlyA so that the export tag can be removed easily. It has then two stop codons, and a terminator at the end.
dTomato (inc RBS, NahR/Psal promoter system)
Regulatory NahR construct with RBS(B0034) and dTomato Red Fluorescent Protein as a reporter.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
ModLac laccase with His-tag and HlyA export tag
The modified CueO was synthesized with iGEM prefix and suffix, RBS(BBa_B0034) and a M-scar after the RBS in order to guarantee the function of the RBS. Two Nsil restriction sites were put before and after a 6xHis-tag so that the enzyme could be purified with an IMAC and so that the tag could be removed. The gene is followed by a KpnI restriction site with glycines and serines on each side, which are small amino acids which increase the flexibility of the protein. This is needed for the export tag HlyA , to make it easier for the resulting signal protein to be folded properly. A second KpnI restriction site is put after the HlyA so that the export tag can be removed easily. It has then two stop codons, and a terminator at the end.
It was derived from a monomeric mutant of the anthozoan coral RFP, DsRed
The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use ??? especially in self-made fluorometry tests ??? because the excitation wavelengths and the emission wavelengths don't overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. Also, its emission peak is at 581 nm and its 50% emission at 629 nm (Figure 1). Characterization has been made with the Anderson promoters BBa_J23101 and (construct BBa_K1688010) and BBa_J23110 (construct BBa_K1688011).
dTomato was derived from a monomeric mutant of the anthozoan coral RFP, DsRed.
The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use ??? especially in self-made fluorometry tests ??? because the excitation wavelengths and the emission wavelengths do not overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. The emission peak is at 581 nm and the 50% emission peak is at 629 nm.
Conversion of the iGEM parts registry to SBOL2.1