Regulatory

Part:BBa_J23119

Designed by: John Anderson   Group: iGEM2006_Berkeley   (2006-08-24)

constitutive promoter family member

BerkiGEM2006-PromotersEppendorfs.jpg
BerkiGEM2006-Promoters.jpg

 Variant RFP (au)
 J23112           1
 J23103           17
 J23113           21
 J23109           106
 J23117           162
 J23114           256
 J23115           387
 J23116           396
 J23105           623
 J23110           844
 J23107           908
 J23106           1185
 J23108           1303
 J23118           1429
 J23111           1487
 J23101           1791
 J23104           1831
 J23102           2179
 J23100           2547
PBca1020-r0040.jpg

Constitutive promoter family
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. J23119 is the "consensus" promoter sequence and the strongest member of the family. All parts except J23119 are present in plasmid J61002. Part J23119 is present in pSB1A2. This places the RFP downstream of the promoter. Reported activities of the promoters are given as the relative fluorescence of these plasmids in strain TG1 grown in LB media to saturation. See part BBa_J61002 for details on their use.

These promoter parts can be used to tune the expression level of constitutively expressed parts. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification. JCAraw


Usage and Biology

iGEM16-CLSB-UK:The consensus promoter Part:BBa_J23119 was assembled with AmilCP blue-purple chromoprotein Part:BBa_K592025. The construct Part:BBa_K2078002 was carried on the pSB1C3 plasmid for expression and amplification in E-Coli.

Pellet from 5ml LB broth
Transformed E-Coli streak plate after 24hours

GreatBay_China 2018:
Team GreatBay_China 2018 characterized J23119, Part:BBa_J23105, and Part:BBa_J23101 by assembling them with Part:BBa_B0034 and a sfGFPPart:BBa_I746916 on three vectors: pUC20 (copy number about 500/cell), pR6K (copy number about 15/cell), pSC101 (copy number about 2/cell). Then we measured the fluorescence by Flow Cytometry as a reference for the TALE stabilized promoter library.
T--GreatBay China--cons.png

The result indicate that the strength of J23119, J23105, and J23101 are about the same as described by team iGEM2006_Berkeley, and the fluorescence increases as the copy number of the vector increases


GreatBay_China have made two improved parts based on J23119.
UP119 is modified by addition of an up-element. (Part:BBa_K2753055)
TALE2 sp6 is a TALE stabilized variant of J23119. (Part:BBa_K2753023)


Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]




>Internal Priming Screening Characterization of BBa_J23119: Has no possible internal priming sites between this BioBrick part and the VF2 or the VR primer.

The 2018 Hawaii iGEM team evaluated the 40 most frequently used BioBricks and ran them through an internal priming screening process that we developed using the BLAST program tool. Out of the 40 BioBricks we evaluated, 10 of them showed possible internal priming of either the VF2 or VR primers and sometime even both. The data set has a range of sequence lengths from as small as 12 bases to as large as 1,210 bases. We experienced the issue of possible internal priming during the sequence verification process of our own BBa_K2574001 BioBrick and in the cloning process to express the part as a fusion protein. BBa_K2574001 is a composite part containing a VLP forming Gag protein sequence attached to a frequently used RFP part (BBa_E1010). We conducted a PCR amplification of the Gag-RFP insert using the VF2 and VR primers on the ligation product (pSB1C3 ligated to the Gag + RFP). This amplicon would serve as template for another PCR where we would add the NcoI and BamHI restriction enzyme sites through new primers for ligation into pET14b and subsequent induced expression. Despite gel confirming a rather large, approximately 2.1 kb insert band, our sequencing results with the VR primer and BamHI RFP reverse primer gave mixed results. Both should have displayed the end of the RFP, but the VR primer revealed the end of the Gag. Analysis of the VR primer on the Gag-RFP sequence revealed several sites where the VR primer could have annealed with ~9 - 12 bp of complementarity. Internal priming of forward and reverse primers can be detrimental to an iGEM project because you can never be sure if the desired construct was correctly inserted into the BioBrick plasmid without a successful sequence verification.


RBS

After an attempt to clone this promoter upstream of the Citrine reporter gene yielded poor expression, the Yale iGEM teaminspected the Ribosome Binding Site used with the promoters and realized that it would need to be improved. We used the Salis Lab Ribosome Binding Site Calculator to design a stronger Ribosome Binding Site.

Yale-2016-anderson-seq.png

Below, we demonstrate that the newly designed Ribosome Binding Site is appropriately compatible with the Anderson Promoter collection.

Yale-2016-anderson-graph-1.png
Yale-2016-anderson-graph-2.png
[edit]
Categories
//chassis/prokaryote/ecoli
//direction/forward
//promoter/anderson
//regulation/constitutive
//rnap/prokaryote/ecoli/sigma70
Parameters
negative_regulators
positive_regulators