Difference between revisions of "Part:BBa K1316009"
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mKate2 is a far-red fluorescent protein used with reporting purposes. mKate2 codon usage is optimised for high expression in mammalian cells (humanised). It is, nevertheless, suitable for propagation in ''E. coli''. The promoters of the ''yqjF'' and ''ybiJ'' genes are activated by the presence of several nitrogen-based compounds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB. Having both promoters in the same plasmid aims to improve the cellular response in front of the inducing chemical compounds (2,4,6-TNT, 2,4-DNT and 1,3-DNB).
 
mKate2 is a far-red fluorescent protein used with reporting purposes. mKate2 codon usage is optimised for high expression in mammalian cells (humanised). It is, nevertheless, suitable for propagation in ''E. coli''. The promoters of the ''yqjF'' and ''ybiJ'' genes are activated by the presence of several nitrogen-based compounds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB. Having both promoters in the same plasmid aims to improve the cellular response in front of the inducing chemical compounds (2,4,6-TNT, 2,4-DNT and 1,3-DNB).
  
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<html>
 
<h3>Characterisation</h3>
 
<h3>Characterisation</h3>
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<p>
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Different type of experiments were conducted to characterize the Landmine Detection BioBricks.
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</p>
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<br>
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<p>
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The different constructs used are:
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<ul>
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<li> p[F]::mKATE, also referred here as LD2  </li>
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<li> p[J]::mKATE, also referred here as LD3  </li>
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<li> p[F] incl. N-enzymes, also referred here  as LD4  </li>
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<li> p[J] incl. N-enzymes, also referred here as LD5  </li>
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<li> p[F]::mKATE p[J]::mKATE, also referred here  as LD6  </li>
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</ul>
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</p>
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<br>
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<h3> Plate Reader </h3>
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<p>
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Using the different final Landmine detection constructs LD2-6, different concentrations of 2,4-DNT were tested (figure 1). The fact that the positive control (constitutively expressed mKate2) presents a clear fluorescence signal at 0 and 50 mg/L, but not at 100 mg/L indicates that high concentrations of 2,4-DNT are toxic for cells after several hours. The toxic compound seems to be 2,4-DNT and not acetonitrile because the sample at 0 mg/L DNT has the same acetonitrile concentration as the 100 mg/L DNT sample. Constructs LD2, LD3 and LD6 as well as LD4 and LD5 with no induction of the N-genes show no clear mKate2 induction over time. Not many conclusions can be drawn form LD2 due to the big standard deviation. However, when the N-genes, constructs LD4 and LD5 showed a clear increase in fluorescence over time for a concentration of 50 mg/L 2,4-DNT. In this situation, besides, LD5 seems to be more sensitive, as there is less leakage (there is less fluorescence signal at 0 mg/L DNT).
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 +
</p>
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<br>
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From this data we concluded that the two best BioBricks for Landmine detection are (in this order) LD5 (p[J] incl. N-enzymes) and LD4 (p[F] incl. N-enzymes), and to obtain a good result the N-genes must be expressed.
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<figure>
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<img src="https://static.igem.org/mediawiki/2014/b/bf/TUDelft_2014_Fluorescence_LD_final.png" width="100%" height="100%">
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<p>
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<figcaption>
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Figure 1: Fluorescence signal measured on the plate reader. Positive control: constitutively expressed mKate2. Negative control: Empty cells carrying no BioBrick
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</figcaption>
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</figure>
  
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The characterisation of this part unfortunately showed no clear improvement in the response of the Landmine detection promoters yqjF and ybiJ (Data not shown).
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<p>
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For more information about the characterisation of this construct visit the <a href="http://2014.igem.org/Team:TU_Delft-Leiden/WetLab/landmine/characterisation"> <b> Landmine Detection module characterisation  </b> </a> on our wiki page!
  
  
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</p>
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</html>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 11:56, 16 October 2014

yqjF promoter coupled to mKate2 reporter gene and ybiJ promoter coupled to mKate2 reporter gene

mKate2 is a far-red fluorescent protein used with reporting purposes. mKate2 codon usage is optimised for high expression in mammalian cells (humanised). It is, nevertheless, suitable for propagation in E. coli. The promoters of the yqjF and ybiJ genes are activated by the presence of several nitrogen-based compounds such as 2,4,6-TNT, 2,4-DNT and 1,3-DNB. Having both promoters in the same plasmid aims to improve the cellular response in front of the inducing chemical compounds (2,4,6-TNT, 2,4-DNT and 1,3-DNB).

Characterisation

Different type of experiments were conducted to characterize the Landmine Detection BioBricks.


The different constructs used are:

  • p[F]::mKATE, also referred here as LD2
  • p[J]::mKATE, also referred here as LD3
  • p[F] incl. N-enzymes, also referred here as LD4
  • p[J] incl. N-enzymes, also referred here as LD5
  • p[F]::mKATE p[J]::mKATE, also referred here as LD6


Plate Reader

Using the different final Landmine detection constructs LD2-6, different concentrations of 2,4-DNT were tested (figure 1). The fact that the positive control (constitutively expressed mKate2) presents a clear fluorescence signal at 0 and 50 mg/L, but not at 100 mg/L indicates that high concentrations of 2,4-DNT are toxic for cells after several hours. The toxic compound seems to be 2,4-DNT and not acetonitrile because the sample at 0 mg/L DNT has the same acetonitrile concentration as the 100 mg/L DNT sample. Constructs LD2, LD3 and LD6 as well as LD4 and LD5 with no induction of the N-genes show no clear mKate2 induction over time. Not many conclusions can be drawn form LD2 due to the big standard deviation. However, when the N-genes, constructs LD4 and LD5 showed a clear increase in fluorescence over time for a concentration of 50 mg/L 2,4-DNT. In this situation, besides, LD5 seems to be more sensitive, as there is less leakage (there is less fluorescence signal at 0 mg/L DNT).


From this data we concluded that the two best BioBricks for Landmine detection are (in this order) LD5 (p[J] incl. N-enzymes) and LD4 (p[F] incl. N-enzymes), and to obtain a good result the N-genes must be expressed.

Figure 1: Fluorescence signal measured on the plate reader. Positive control: constitutively expressed mKate2. Negative control: Empty cells carrying no BioBrick

For more information about the characterisation of this construct visit the Landmine Detection module characterisation on our wiki page!

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 128
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1488
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 733
    Illegal BsaI.rc site found at 922
    Illegal BsaI.rc site found at 1993
    Illegal BsaI.rc site found at 2182