Coding

Part:BBa_K2560033

Designed by: Tobias Hensel   Group: iGEM18_Marburg   (2018-09-14)

Phytobrick version of BBa_E1010

This is the Phytobrick version of the coding sequence BBa_E1010 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

Open reading frames (ORFs) are sequences that can theoretically be translated into functional proteins. They are predicted by a start codon (ATG) at their beginning and a stop codon (TAA, TAG, TGA) at the end separated by multiple codons. When an ORF is confirmed to code for a functional protein it is classified as a coding sequence (CDS). CDSs code only proteins. To translate them they need a promoter upstream the start codon.A terminator downstream of the sequence end the translation. In prokaryotes an RBS sequence is needed between the promoter and the start codon. In eukaryotes CDSs are flanked by untranslated regions (UTRs). In addition eukaryotic CDSs contain introns which are removed after transcription by splicing to form the mature mRNA which is translated into the functional protein outside the nucleus.

Characterization

Figure 1: Mean ratio of reporter signal over medium blank during the coarse of the experiment.




After having established a reliable workflow for V. natriegens, we investigated four different reporters and measured the signal to blank ratio. Test constructs (shown in figure 2) were built by using the same set of parts except for the coding sequence. sfGFP, RFP, YFP and the lux operon were analyzed for their performance in V. natriegens. The best signal to blank ratio by far was achieved for the lux operon (2000), followed by sfGFP (3), RFP (1) and YFP (no detectable signal). The main explanation for the superior performance of the lux operon is the almost complete absence of background signal without reporter expression. This makes the lux operon a perfect reporter that can even be used to analyze extremely low levels of expression caused by very weak promoters or terminator read through. Based on this finding, we decided to use the lux operon as our reporter for all subsequent experiments.

A
B
C
D
Figure 2: Test constructs for reporter experiment
Plasmids were built with four different reporters.
A) Lux B) RFP C) sfGFP D) YFP

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 552
    Illegal AgeI site found at 664
  • 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