Difference between revisions of "Part:BBa K1468000"

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===Part Stability===
 
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The standard conditions for ''E. coli'' growing are 37°C and 200 rpm. Varying these conditions in terms of salinity, pHTemperature, pH, salinity, ultraviolet radiation exposure and vacuum) and measuring the growth after three hours, (O.D.595) as well as BBa_K1468000 (fluorescence), was used to test the effect on BBa_K1468000 expression (all data are the average of at least three measurements; fluorescence values are normalized to OD595).
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The results obtained were:
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https://static.igem.org/mediawiki/2014/6/65/Bb1pLDM2.png
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'''Figure 2. ''' The strain XL1 Blue transformed with a Biobrick which encodes GFP (BBa_K1468000) was grown at different temperatures in a thermocycler for three hours, the cells were collected and O.D600 was measured. Fluorescence was also determined and compared to the non-transformed/wildtype or control strain. As the plot shows, Biobrick expression is affected by growth at different temperatures, fluorescence (normalized to O.D600) is higher around ''E. coli'' optimal growth temperature (37-41 °C); over or below this rather short temperature range fluorescence decreases.
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https://static.igem.org/mediawiki/2014/5/5d/Bb1pLDM3.png
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'''Figure 3. ''' BBa_K1468000 expression under different temperatures, including temperature fluctuation (from 37°C to 41°C, every minute each) in a thermocycler. Controls were incubated at 37 and 41°C.  After an incubation period of 3 hours, O.D.600 alongside fluorescence was measured. The results show that the expression of the Biobrick is not affected by cycling temperatures.
  
 
<html><img src="https://static.igem.org/mediawiki/2014/c/cb/GFPpLDM3.png"  
 
<html><img src="https://static.igem.org/mediawiki/2014/c/cb/GFPpLDM3.png"  

Revision as of 16:01, 13 October 2014

pJ23104 + gene encoding ZsGreen

Contents

Description
Usage and Biology
Universality
Compatibility with current standards and systems (Modularity)
Innovative point
FAQ
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. Varying these conditions in terms of salinity, pHTemperature, pH, salinity, ultraviolet radiation exposure and vacuum) and measuring the growth after three hours, (O.D.595) as well as BBa_K1468000 (fluorescence), was used to test the effect on BBa_K1468000 expression (all data are the average of at least three measurements; fluorescence values are normalized to OD595).

The results obtained were:

Bb1pLDM2.png

Figure 2. The strain XL1 Blue transformed with a Biobrick which encodes GFP (BBa_K1468000) was grown at different temperatures in a thermocycler for three hours, the cells were collected and O.D600 was measured. Fluorescence was also determined and compared to the non-transformed/wildtype or control strain. As the plot shows, Biobrick expression is affected by growth at different temperatures, fluorescence (normalized to O.D600) is higher around E. coli optimal growth temperature (37-41 °C); over or below this rather short temperature range fluorescence decreases.

Bb1pLDM3.png

Figure 3. BBa_K1468000 expression under different temperatures, including temperature fluctuation (from 37°C to 41°C, every minute each) in a thermocycler. Controls were incubated at 37 and 41°C. After an incubation period of 3 hours, O.D.600 alongside fluorescence was measured. The results show that the expression of the Biobrick is not affected by cycling temperatures.

Compatibility with current standards and systems (Modularity)

Innovative point

FAQ

Further reading and additional information

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]