Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Malcolm Campbell
Date created: 2012-12-13 12:00:00
Date modified: 2015-08-31 04:08:22
For Testing New Promoters via Golden Gate Assembly v2
| Types | DnaRegion |
| Roles | engineered_region Reporter |
| Sequences | BBa_J100104_sequence (Version 1) |
Description
J100104 was built by Todd Eckdahl and Malcolm Campbell. This part allows users to clone and test new promoters without gel purification or other preparation of DNA - it is ideal for teaching labs. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and Ligase. A new promoter can be derived from synthetic oligos, PCR, or a plasmid clone. The new promoter must be flanked by BsaI sites that produce the 4 nt overhangs required for assembly (see Part:BBa_J119022 as an example, or the picture below). Successful GGA assembly replaces a reporter gene that turns E. coli blue (J100103) with the new promoter. Upon assembly, a functional new promoter should cause RFP expression. J100104 and its sister part Part:BBa_J100091 incorporate the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.Below is a picture of the portion that pops out when digested with Bsa I. J100103rev represents the reverse complement of J100103 and is designed to be used for Golden Gate Assembly to swap out the blue protein production for a promoter of your choosing. This can be done by simply mixing J100104 with oligos that self-assemble into dsDNA with compatible sticky ends. J100104 and its sister part Part:BBa_J100091 are ideal for undergraduates in a teaching lab to discover and characterize new promoters for use in synthetic biology.
See the full GGA protocol online.
[[Image:J100028.png]]
Notes
J100104 contains BBa biobrick prefix + 4 base overhang + BsaI site cutting to the left + reverse complement of blue reporter protein [[Part:BBa_J100103]] + BsaI site that cuts to the right + 4 base overhang + BD18 bicistronic RBS for more reliable translation ([[Part:BBa_J119024]]) + mRFP [[Part:BBa_E1010]] + BBa biobrick suffix.Source
Built from PCR of J100103 and J100091 on modified version of pSB1A2 that has its BsaI site removed from the AmpR gene.| Sequence Annotation | Location | Component / Role(s) |
| BBa Prefix right sticky end BsaI cuts left J100103 rev BsaI cuts right left sticky end BD18 mRFP double stop double stop BBa suffix blue protein BD18 reverse P2 reverse | 1,22 23,26 28,33 34,833 834,839 841,844 845,929 930,1610 1605,1610 34,39 1611,1631 34,702 703,787 788,833 | engineered_region feature/BioBrick feature/dna sequence_feature feature/binding non_covalent_binding_site CDS feature/cds sequence_feature feature/dna sequence_feature feature/dna feature/rbs ribosome_entry_site feature/cds CDS feature/stop stop_codon feature/stop stop_codon engineered_region feature/BioBrick feature/protein CDS ribosome_entry_site feature/rbs promoter feature/promoter |