Difference between revisions of "Part:BBa K2560034"

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[[File:T--Marburg--Terminator_Construct.png|500px|thumb|left|'''Figure 1''': <b> Terminator test construct. </b> <br> LVL2 plasmids were created for these experiments consisting of a RFP transcription unit with the strong constitutive promoter J23100, followed by the <i>lux</i> operon with the promoter dummy. The terminator located at the 3' end of the RFP transcription unit is the part which is characterized in this experiment.]]
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[[File:T--Marburg--Terminator_Construct.png|400px|thumb|left|'''Figure 1''': <b> Terminator test construct. </b> <br> LVL2 plasmids were created for these experiments consisting of a RFP transcription unit with the strong constitutive promoter J23100, followed by the <i>lux</i> operon with the promoter dummy. The terminator located at the 3' end of the RFP transcription unit is the part which is characterized in this experiment.]]
  
 
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Revision as of 20:24, 16 October 2018


Phytobrick version of BBa_B0010

This is the Phytobrick version of the Terminator BBa_B0010 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.

Overview

Terminators are sequences downstream CDSs promoting dissociation of the transcriptional complex. In prokaryotes two kind of transcription terminators are reported. The first ones are Rho-independent terminators. This sequences build up hairpin structures during transcription disrupting the transcriptional complex. The mechanism of disruption is hypothesized to occur through allosteric effects of the hairpin binding and competitive kinetics. The other group are Rho-dependent terinators which require Rho and ATP. This terminators are found in bacteria and phages. The Rho factor binds to a cytosine-rich sequence on the mRNA where it hydrolyzes ATP, translocates down the mRNA and stimulates the dissociation of the transcription complex contact is established.

Characterization

The Marburg Collection contains five terminators plus one terminator dummy that can be used as a placeholder. To obtain experimental data for this category of parts, we built a set of terminator test constructs to measure the extent of transcriptional readthrough and therefore the strength of a terminator. The terminator test constructs are built as LVL2 plasmid with our toolbox. The strongest constitutive promoter J23100 drives the expression of RFP which is the first transcription unit. The Lux operon is placed downstream with the promoter dummy instead of an active promoter. Both transcription units are separated by the terminator, which is the focus of this characterization, downstream of the RFP CDS.


Figure 1: Terminator test construct.
LVL2 plasmids were created for these experiments consisting of a RFP transcription unit with the strong constitutive promoter J23100, followed by the lux operon with the promoter dummy. The terminator located at the 3' end of the RFP transcription unit is the part which is characterized in this experiment.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 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)