Regulatory

Part:BBa_K2560007

Designed by: Tobias Hensel   Group: iGEM18_Marburg   (2018-08-20)
Revision as of 20:25, 11 October 2018 by Henselt (Talk | contribs) (→‎Marburg Toolbox)


Phytobrick version of BBa_J23100

This is the Phytobrick version of the promoter BBa_J23100 and was build as a part of the Marburg Collection. Instructions of how to use the Marburg Collection are provided at the bottom of the page.

T--Marburg--LUXPicture new.png


BerkiGEM2006-Promoters.jpg


 Variant Lux (au)
 
 K2560131 (Dummy)    0.025
 K2560019 (J23103)   0.032
 K2560026 (J23113)   0,038
 K2560023 (J23109)   0,052
 K2560009 (J23104)   0,058
 K2560029 (J23117)   0,090
 K2560025 (J23111)   0,098
 K2560028 (J23116)   0,134
 K2560021 (J23107)   0,136
 K2560027 (J23114)   0,163
 K2560024 (J23110)   0,169
 K2560018 (J23102)   0,245
 K2560030 (J23118)   0,348
 K2560020 (J23105)   0,387
 K2560015 (J23115)   0,398
 K2560014 (J23106)   0,502
 K2560017 (J23101)   0,510
 K2560022 (J23108)   0,768
 K2560007 (J23100)   1
Figure 1. Relative promoter strenghts of promoters from the Anderson Collection in Vibrio natriegens. The promoter test construct is shown in Figure 2. Contruct were messured in quadruplicates and in three indipendent experiments.
Figure 2. Contruct for messuring promoter strenghts. The label "Tested Promoters" is a place holder for the respective promoter. The plasmid was assembled from eight basic parts by Golden Gate Assembly.



Usage and Biology

This is the Phytobrick version of the promoter BBa_J23100. We build up this part via de novo synthesis by annealing the single-stranded complementary oligonucleotides aiGEM1009_J23100_fwd and aiGEM1010_J23100_rev to form the double-stranded Anderson Promotor BBa_J23100. If you stuggle with de novo synthesis we recomended this site on the iGEM Wiki.
Marburg 2018 characterized this part in Vibrio natriegens using the lux operon of Photorhabdus luminescens (BBa_K2560051). The messured relative promotor strenghts in Vibrio natriegens were correlated to the relative promotor strenghts in E. coli (iGEM Berkeley 2006). The parts sequence was verified by Sanger sequencing.


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]


Marburg Toolbox

We proudly present the Marburg Collection, a novel golden-gate-based toolbox containing various parts that are compatible with the PhytoBrick system and MoClo. Compared to other bacterial toolboxes, the Marburg Collection shines with superior flexibility.We overcame the rigid paradigm of plasmid construction - thinking in fixed backbone and insert categories - by achieving complete de novo assembly of plasmids.

36 connectors facilitate flexible cloning of multigene constructs and even allow for the inversion of individual transcription units. Additionally, our connectors function as insulators to avoid undesired crosstalk.

The Marburg Collection contains 122 parts in total, including:
inducible promoters, reporters, fluorescence and epitope tags, oris, resistance cassettes and genome engineering tools.To increase the value of the Marburg Collection, we additionally provide detailed experimental characterization for V. natriegens and a supportive software. We aspire availability of our toolbox for future iGEM teams to empower accelerated progression in their ambitious projects.


Figure 3: Hierarchical cloning is facilitated by subsequent Golden Gate reactions. Basic building blocks like promoters or terminators are stored in level 0 plasmids. Parts from each category of our collection can be chosen to built level 1 plasmids harboring a single transcription unit. Up to five transcription units can be assembled into a level 2 plasmid.
Figure 4: Additional bases and fusion sites ensure correct spacing and allow tags. Between some parts, additional base pairs were integrated to ensure correct spacing and to maintain the triplet code. We expanded our toolbox by providing N- and C- terminal tags by creating novel fusions and splitting the CDS and terminator part, respectively.


Parts of the Marburg Toolbox




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