![](https://parts.igem.org/images/partbypart/icon_terminator.png)
Part:BBa_K2560034
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.
![](/wiki/images/8/83/T--Marburg--Terminator_Construct.png)
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
![](/wiki/images/2/29/T--Marburg--Cloning_Overview.png)
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.
![](/wiki/images/4/4d/T--Marburg--Overview_Marburg_Toolbox.png)
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
![](/wiki/images/2/25/T--Marburg--5%27Con.png)
- 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)
![](/wiki/images/c/cc/T--Marburg--Promotor.png)
- 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)
![](/wiki/images/9/9c/T--Marburg--3%27Con.png)
- 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)
![](/wiki/images/e/ee/T--Marburg--Antibiotic_Resistance.png)
- 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
None |