Regulatory

Part:BBa_K2560007

Designed by: Tobias Hensel   Group: iGEM18_Marburg   (2018-08-20)
Revision as of 16:15, 14 October 2018 by Henselt (Talk | contribs)


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.

Promoters

Promoters are genetic modules were the RNA polymerase is recruited to start RNA transcription. They are divided in two groups: constitutive promoters which transcribe RNA permanently and inducible promoters which start the transcription as a response to a stimulus. Inducible promoters can be regulated by transcription activation or repression. To start the RNA transcription the RNA polymerase complex is not sufficient. Therefore sigma70 factors are required. The sigma70 factor binds to the Pribnow box – two motifs -10 and -35bp upstream the CDS – recruiting the RNA transcrition complex enabling the transcription. Based on this knowledge a collection of synthetic constitutive promoters have been developed by Chris Anderson and made available on the iGEM repository. Sigma70 factors are not the only ones inducing RNA transcription. A lot of research is currently make on ECFs (extracytoplasmic function). This factors bind the promoter regions and are crucial for the recruitment of the RNA polymerase just like Sigma70 factors. Nevertheless ECFs can be bound highly specific by membrane bound anti-sigma factors inhibiting the RNA transcription. This makes them very interesting for synthetic biology for the design and control of genetic circuits.

Characterisation

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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


We assembled 19 test plasmids with golden-gate-assembly and measured their expression strength, following our selfmade workflows. The results are shown in Figure 1. We observed an even distribution of the tested promoters throughout the dynamic range. The strongest promoter K2560007 (J23100) yielded 40 fold stronger signal than the promoter dummy and was used as a reference to calculate relative promoter strengths. The test constructs were built with dummy connectors which did not possess insulator elements. We assume that this resulted in additional expression caused by transcription throughout the rest of the plasmid, e.g. ori and antibiotic resistance. This is thought to add the same extent of signal to all measured promoters thus reducing the overall dynamic range. To further evaluate this assumption, we could repeat this experiment with one of our insulators instead of the dummy connector.

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

Marburg 2018 characterized this part in Vibrio natriegens using the lux operon of Photorhabdus luminescens (BBa_K2560051). The messured relative promoter strenghts in Vibrio natriegens were correlated to the relative promoter 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 123 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




Tags and Entry Vectors




  • K2560001 (Entry Vector with RFP dropout)
  • K2560002 (Entry Vector with GFP dropout)
  • K2560005 (Resistance Entry Vector with RFP Dropout)
  • K2560006 (Resistance Entry Vector with GFP Dropout)
  • K2560305 (gRNA Entry Vector with GFP Dropout)
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Categories
Parameters
None