Reporter

Part:BBa_K1468000

Designed by: Pedro Luis Dorado Morales   Group: iGEM14_Valencia_Biocampus   (2014-10-03)

pJ23104 + gene encoding ZsGreen

Contents

Description
Usage and Biology
Universality
Compatibility with current standards and systems (Modularity)
Innovative point
Doubts and FAQs
Further reading and additional information

Description

This part consists of the ZsGreen coding sequence of Zoanthus spp., placed under the control of the constitutive promoter J23104 -promoter designed by J. Anderson (iGEM 2006, Berkeley). This promoter belongs to a family of constitutive promoter parts isolated from a small combinatorial library.

Usage and Biology

The biobrick promoter J23104, as it was mentioned above, is constitutive. It was tested by two groups of iGEMites, additonally to Anderson's: 2012 iGEM Team Göttingen (by C. Krüger and J. Kampf) and iGEM Cinestav (National Autonomous University of Mexico & National Polytechnic Institute; Center for Research and Advanced Studies at Irapuato).

Results obtained here: [1]

Universality

A priori, BBa_K1468000 can be used in a wide variety of prokariotic and eukariotic cells. However, some considerations should be taken into account:

- The J23104 promoter, constitutive, has been tested just in Escherichia coli strains. This fact ensures its correct operation in a group of closed related species but not to the wide range of microorganism.

- The ZsGreen encoding gene sequence is optimized forEscherichia coli codon usage. Some codon usage modification must be carried out for non-related organisms.

- There are some limitations related to the plasmid, where the construction is cloned. I mean, the backbones Ori is not universal, so it would be necessary to check that if Ori is compatible with the organism we want to transform.

Part Stability

The standard conditions for E. coli growing are 37°C and 200 rpm in LB medium. Varying these conditions, in terms of salinity, pH and temperature, does the Biobrick behave in the same way?

For answering this question, the XL1 Blue strain was selected and subjected to the following unusual conditions: different salt concentrations (0% p/v, 1% p/v, 2% p/v, 3% p/v), non-usual pHs (5, 6, 7, 8) and a range of temperatures ranging from 34-44.

Results obtained:

Figure 1. Transformed XL1 Blue strain ratio GFP fluorescence/OD595 at different concentrations of salt (NaCl). As it can be observed, Biobrick output is not substantially affected by salt concentrations.

All the salt concentrations refers to the standard composition of LB medium, i.e., 1% means 1% additional to the LB standard formulation.


Figure 2. Transformed XL1 Blue strain ratio GFP fluorescence/OD595 at different pHs. As it can be observed, Biobrick output is not substantially affected by pH.


Figure 3. Transformed XL1 Blue strain ratio GFP fluorescence/OD595 at different temperatures. As it can be observed, Biobrick output is affected by temperature, being higher around E. coli optimal growth temperature (37-41 °C).

Compatibility with current standards and systems (Modularity)

Two BioBricks are considered to behave orthogonal when they interact in a predicted and specific way.

To check the modularity of BBa_K1468000, we co-transform one E. coli strain, XL1Blue, with the following two biological parts: BBa_K1468000 (Bb1) & BBa_K1468001(Bb2) in same plasmid (different resistance cassette: Amp & Kan), under the control of the same promoter and both codifing similar proteins ZsGreen and RFP.


Standard fluorimetry assays and flow cytometry were used to carry out the measures.


The results obtained were:


Caption. BBa_K1468000 (Bb1), BBa_K1468001 (Bb2), e.k.p. (empty kanamycin plasmid, no insert), e.a.p. (empty ampicillin plasmid, no insert), wild type (WT).


BBa_K1468000 was cloned in a pUC57Amp. BBa_K1468001 was cloned in a pUC57Kan.

e.k.p, e.a.p and WT are the experimental controls.


Caption: BBa_K1468000 (Bb1), BBa_K1468001 (Bb2), e.k.p. (empty kanamycin plasmid, no insert), e.a.p. (empty ampicillin plasmid, no insert), wild type (WT).


BBa_K1468000 was cloned in a pUC57Amp. BBa_K1468001 was cloned in a pUC57Kan.

e.k.p, e.a.p and WT are the experimental controls.

Innovative point

The BBa_K1468000 part presents a deep characterization in a range of E. coli strains (see experience [2]), conditions (see Part Stability) and assays carried out (see Compatibility with current standards and Systems (Modularity)), which, probably makes of BBa_K1468000 one of the best characterized parts of the Registry.

Doubts and FAQs

For any question, send an e-mail to the address shown below and we will reply you as soon as possible.

vlc.biocampus.igem@gmail.com

Further reading and additional information

Nakamura, Y. et al.Bright fluorescence monitoring system utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for human G-protein-coupled receptor signaling in microbial yeast cells. PLOS ONE (2013). DOI: 10.1371/journal.pone.0082237

Paragraph: Comparison of two green fluorescence proteins (ZsGreen and EGFP) as reporter genes.

Andersen, J.B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Applied and Environmental Microbiology (1998) 64(6), 2240-2246

Tsien, R.Y. The green fluorescent protein. Annual Review of Biochemistry (1998) 67, 509–544

Kelly, J.R. et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering (2009). DOI:10.1186/1754-1611-3-4

Talk about Synthetic Biology: BioBricks & Standardization [3]


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 596
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/reporter
//cds/reporter/gfp
//chassis/multihost
//chassis/prokaryote
//chassis/prokaryote/ecoli
//classic/reporter
//classic/reporter/constitutive
//function/reporter
//function/reporter/fluorescence
//promoter/anderson
//rbs/prokaryote/constitutive/anderson
//regulation/constitutive
//terminator
Parameters
chassisE.coli strains: DH5-alpha, XL1 Blue, JM109, DH10B, HB101, BL21 (DE3)
controlConstitutive promoter: J23104
emissionλem = 505 nm
excitationλex = 493 nm
outputLight
proteinZsGreen