
Part:BBa_K2560042
Phytobrick version of sfGFP
This is the Phytobrick version of the coding sequence sfGFP 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
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.




Plasmids were built with four different reporters.
A) Lux B) RFP C) sfGFP D) YFP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 10
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.

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.

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

- K2560011 (5'Connector Dummy)
- K2560055
(1-6
Connector) - K2560065 (5'Con1)
- K2560066 (5'Con2)
- K2560067 (5'Con3)
- K2560068 (5'Con4)
- K2560069 (5'Con5)
- K2560075 (5'Con1
Short Res) - K2560076 (5'Con2
Short) - K2560077 (5'Con3
Short) - K2560078 (5'Con4
Short) - K2560079 (5'Con5
Short) - K2560095 (5'Con1 inv)
- K2560096 (5'Con2 inv)
- K2560097 (5'Con3 inv)
- K2560098 (5'Con4 inv)
- K2560099 (5'Con5 inv)
- K2560105 (5'Con5 inv
Ori) - K2560107 (5'Con1
Res)

- K2560007 (J23100)
- K2560009 (J23104)
- K2560014 (J23106)
- K2560015 (J23115)
- K2560017 (J23101)
- K2560018 (J23102)
- K2560019 (J23103)
- K2560020 (J23105)
- K2560021 (J23107)
- K2560022 (J23108)
- K2560023 (J23109)
- K2560024 (J23110)
- K2560025 (J23111)
- K2560026 (J23113)
- K2560027 (J23114)
- K2560028 (J23116)
- K2560029 (J23117)
- K2560030 (J23118)
- K2560031 (J23119)
- K2560123
(pTet) - K2560124 (pTrc)
- K2560131 (Promoter Dummy)

- K2560012 (3'Connector Dummy)
- K2560070 (3'Con1)
- K2560071 (3'Con2)
- K2560072 (3'Con3)
- K2560073 (3'Con4)
- K2560080 (3'Con5 Ori)
- K2560100 (3'Con1 inv
Short) - K2560101 (3'Con2 inv
Short) - K2560102 (3'Con3 inv
Short) - K2560103 (3'Con4 inv
Short) - K2560104 (3'Con5 inv
Short) - K2560106 (3'Con1 inv
Short Res) - K2560108 (3'Con1 inv)
- K2560109 (3'Con1 inv
Res) - K2560110 (3'Con2 inv)
- K2560111 (3'Con3 inv)
- K2560112 (3'Con4 inv)
- K2560113 (3'Con5 inv)

- K2560048 (Cam. Res. RFP)
- K2560056
(Kan. Res. (pSB3K3) RFP) - K2560057
(Kan. Res. (pSB3K3) GFP) - K2560058
(Tet. Res. (pSB3T5) RFP) - K2560059
(Tet. Res. (pSB3T5) GFP) - K2560125 (Carb. Res. RFP)
- K2560126 (Carb. Res. GFP)
- K2560127 (Carb. Res. into BBa_K2560002)
- K2560132 (Cam. Res. into BBa_K2560002)
- K2560133
(Kan. Res. into BBa_K2560002) - K2560134
(Tet. Res. into BBa_K2560002)
Tags and Entry Vectors
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