Difference between revisions of "Part:BBa K2505009"

 
 
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This part produces GFP induced by C8.
 
  
 
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<partinfo>BBa_K2505009 parameters</partinfo>
 
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This part produces GFP induced by 3OC8AHL molecules ([N-]acyl-homoserine lactones, AHLs), 3OC8 HSL (hereafter C8).
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<html>
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</p>
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</html>
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__TOC__
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==Characterization==
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In our project, we use a kind of Quorum Sensing signaling molecules ([N-]acyl-homoserine lactones, AHLs), 3OC8 HSL (hereafter C8) as a signaling molecule of inter-kingdom communication from bacteria to human cells. Therefore, we searched the way to detect that C8 was synthesized by <i>E. coli</i> introduced C8 synthesis gene, <i>traI</i>. To evaluate Tra system (a kind of Quorum Sensing derived from Agrobacterium tumefaciens), our team constructed the plasmids that constitutively express TraR (C8 reporter protein) and, C8-TraR inducible promoter, Ptra and subsequence <i>gfp</i>.
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==Result==
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The results showed that the value of RFU of GFP / Turbidity of samples and that of negative controls were same. This indicated that GFP expression was not induced by TraR-C8 complex.
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[[File:T--TokyoTech--TraR reporter assay.png|thumb|left|600px| '''Figure 1:''' '''RFU of GFP / Turbidity of TraR Reporter Assay'''<br style="clear: both" /> ]]<br>
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<br style="clear: both" />
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==Discussion==
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From the results, Tra reporter system did not work in <i>E. coli</i> even though the temperature conditions were changed. This is because the tra box where TraR-C8 complex binds upstream Ptra is essential to active transcription and the number of this region is not enough for non-A. tumefaciens creatures to activate transcription.
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==Material and Method==
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===Plasmids===
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*Sample
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<span style="margin-left: 20px;">Ptet – rbs – <i>traR</i> (pSB6A1)
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<span style="margin-left: 20px;">Ptra – rbs – <i>gfp</i> (pSB3K3)
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*Negative control
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<span style="margin-left: 20px;">pSB6A1
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<span style="margin-left: 20px;">pSB3K3
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===Construction===
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*Strain
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<span style="margin-left: 20px;">All the plasmids were prepared in <i>E. coli</i> DH5a strain.
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*Medium
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<span style="margin-left: 20px;">LB medium A:LB medium containing ampicillin (50 µg/ mL) and kanamycin (50 µg/ mL).
 +
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===Assay Protocol===
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The following experiments are performed at 37˚C unless otherwise stated.
 +
 +
1. Prepare overnight cultures for each sample in 3 mL LB medium A at 37˚C with vigorous shaking.
 +
 +
2. Dilute the overnight cultures to 1 / 60 in fresh LB medium A (1.2 mL).
 +
 +
3. Incubate the fresh cultures for 1 h at 37˚C or 30˚C with vigorous shaking.
 +
 +
4. Add 80 mM C8 or DMSO to each 800 µL sample at the final concentration 20 µM.
 +
 +
5. Incubate the samples for 4 h at 37˚C or 30˚C with vigorous shaking.
 +
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6. Add 100 µL of the samples to each well of a plate reader.
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7. Measure RFU of GFP at 490 nm as an excitation wavelength, 525 nm as an emission wavelength. 
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8. Measure the turbidity at 600 nm.
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==Reference==
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C Fuqua, S C Winans. (1996) Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J Bacteriol. 178: 435–440.

Latest revision as of 18:40, 1 November 2017

Ptra-rbs-gfp-tt

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 105
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 821


This part produces GFP induced by 3OC8AHL molecules ([N-]acyl-homoserine lactones, AHLs), 3OC8 HSL (hereafter C8).


Characterization

In our project, we use a kind of Quorum Sensing signaling molecules ([N-]acyl-homoserine lactones, AHLs), 3OC8 HSL (hereafter C8) as a signaling molecule of inter-kingdom communication from bacteria to human cells. Therefore, we searched the way to detect that C8 was synthesized by E. coli introduced C8 synthesis gene, traI. To evaluate Tra system (a kind of Quorum Sensing derived from Agrobacterium tumefaciens), our team constructed the plasmids that constitutively express TraR (C8 reporter protein) and, C8-TraR inducible promoter, Ptra and subsequence gfp.

Result

The results showed that the value of RFU of GFP / Turbidity of samples and that of negative controls were same. This indicated that GFP expression was not induced by TraR-C8 complex.

Figure 1: RFU of GFP / Turbidity of TraR Reporter Assay


Discussion

From the results, Tra reporter system did not work in E. coli even though the temperature conditions were changed. This is because the tra box where TraR-C8 complex binds upstream Ptra is essential to active transcription and the number of this region is not enough for non-A. tumefaciens creatures to activate transcription.

Material and Method

Plasmids

  • Sample

Ptet – rbs – traR (pSB6A1)

Ptra – rbs – gfp (pSB3K3)

  • Negative control

pSB6A1

pSB3K3

Construction

  • Strain

All the plasmids were prepared in E. coli DH5a strain.

  • Medium

LB medium A:LB medium containing ampicillin (50 µg/ mL) and kanamycin (50 µg/ mL).

Assay Protocol

The following experiments are performed at 37˚C unless otherwise stated.

1. Prepare overnight cultures for each sample in 3 mL LB medium A at 37˚C with vigorous shaking.

2. Dilute the overnight cultures to 1 / 60 in fresh LB medium A (1.2 mL).

3. Incubate the fresh cultures for 1 h at 37˚C or 30˚C with vigorous shaking.

4. Add 80 mM C8 or DMSO to each 800 µL sample at the final concentration 20 µM.

5. Incubate the samples for 4 h at 37˚C or 30˚C with vigorous shaking.

6. Add 100 µL of the samples to each well of a plate reader.

7. Measure RFU of GFP at 490 nm as an excitation wavelength, 525 nm as an emission wavelength.

8. Measure the turbidity at 600 nm.

Reference

C Fuqua, S C Winans. (1996) Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J Bacteriol. 178: 435–440.