BBa_J100091

BBa_J100091 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_J100091
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Malcolm Campbell and Todd Eckdahl
Date created: 2012-08-19 11:00:00
Date modified: 2015-08-31 04:08:22

For Testing New Promoters via Golden Gate Assembly



Types
DnaRegion

Roles
Measurement

engineered_region

Sequences BBa_J100091_sequence (Version 1)

Description

The construct allows for the cloning and testing of new promoter sequences. It is a destination vector for Golden Gate Assembly using BsaI and Ligase, based on part [[Part:BBa_J100028]]. 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]]). Assembly replaces the double terminator in the destination vector with the new promoter. Upon assembly, a functional new promoter will be expected to cause RFP expression. The destination vector also incorporates the BD18 bicistronic translational junction (see [[Part:BBa_J119024]]) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.

Notes

The major question is what plasmid to use. Since both [[Part:BBa_J119044]] and [[Part:BBa_J100091]] both contain a transcriptional terminator (TT), this may lead to recombination if more than two TTs are in the same plasmid. We have some evidence that [[Part:BBa_J119044]] can undergo some recombination when cloned into the plasmid pSB1A8 [[Part:BBa_J119043]]. Therefore, we recommend [[Part:BBa_J119044]] and [[Part:BBa_J100091]] be cloned into a modified version of pSB1A2 that has the Bsa I site removed from the ampicillin gene. We call this modified plasmid pSB1A2 BR for Bsa I Removed.

Source

This part was engineered by Todd Eckdahl and Malcolm Campbell. They devised this as part of a national effort to have undergraduates contribute to a registry of functional promoters (RFP; Registry of Functional Promoters). There is a very similar part called J119044 [[Part:BBa_J119044]]. The only difference between these two parts is that J00091 uses E1010 [[Part:BBa_E1010]] version of RFP while [[Part:BBa_J119044]] uses an E. coli-optimized set of codons and was produced by GeneArt.

Sequence Annotation Location Component / Role(s)
BBa Prefix
BsaI sticky end left
BsaI cuts left
TT B0014
BsaI cuts right
BsaI sticky end right
BD18 bicistron
leader RBS
RBS for RFP
Stop for BD18 leader
Start for RFP
RFP E1010
Double Stop for E1010
BBa suffix
PCR For Primer
PCR Rev Primer
1,22
23,26
28,33
34,128
129,134
136,139
140,227
157,165
210,218
223,225
225,227
225,905
900,905
906,926
1,20
232,251
engineered_region feature/BioBrick
feature/dna sequence_feature
feature/misc sequence_feature
feature/stem_loop stem_loop
feature/misc sequence_feature
feature/dna sequence_feature
feature/misc sequence_feature
feature/rbs ribosome_entry_site
feature/rbs ribosome_entry_site
feature/stop stop_codon
start_codon feature/start
CDS feature/protein
feature/stop stop_codon
feature/BioBrick engineered_region
feature/primer_binding primer_binding_site
feature/primer_binding primer_binding_site
igem#experience
Works
 
igem#sampleStatus
Not in stock
igem#status
Unavailable
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_J100091/1