Part:BBa_K1468000

pJ23104 + gene encoding ZsGreen

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

chassis	E.coli strains: DH5-alpha, XL1 Blue, JM109, DH10B, HB101, BL21 (DE3)
control	Constitutive promoter: J23104
emission	λem = 505 nm
excitation	λex = 493 nm
output	Light
protein	ZsGreen