Difference between revisions of "Part:BBa K1992005"

 
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<partinfo>BBa_K1992005 short</partinfo>
 
<partinfo>BBa_K1992005 short</partinfo>
  
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introduction
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<span class='h3bb'>Sequence and Features</span>
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Tar chemoreceptor is one of four native chemoreceptors of E.coli, it is mediate chemotaxis response towards aspartate and away from Ni and Co (&#1502;&#1511;&#1493;&#1512;). This device allows the expression of a functionally native Tar chemoreceptor.
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<partinfo>BBa_K1992005 SequenceAndFeatures</partinfo>
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<br>
  
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usage and biology
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==Introduction==
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This device is very similar to our K1992004 device the only difference is the RBS, here the expression system is using the native RBS of the Tar receptor as found in the E.coli genome (K1992000).  
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Tar chemoreceptor is one of four native chemoreceptors of E.coli, it is mediate chemotaxis response towards aspartate and away from Ni and Co (1). This device allows the expression of a functionally native Tar chemoreceptor.
  
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expriments and results
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==Usage and biology==
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In order to test the device it was cloned to chemoreceptor free strain (UU1250) and tested for chemotaxis ability using swarming assay.
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This device is very similar to our <partinfo>K1992004</partinfo> device it is only differ by its RBS. Here the expression system is using the native RBS of the Tar receptor as found in the E.coli genome (<partinfo>K1992000</partinfo>).
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As can be seen in figure 1 the cells expressing the Tar chemoreceptor create a halo indicating a functional chemotaxis result, which compared to the Tar expression system using a strong RBS(B0034).
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==Expriments and results==
  
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Figure 1
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In order to test the device it was transformed to chemoreceptor free strain (UU1250) and tested for chemotaxis ability using [http://2016.igem.org/Team:Technion_Israel/Experiments swarming assay].
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As can be seen in figure 1 the cells expressing the Tar chemoreceptor create a halo indicating a functional chemotaxis result.The larger radius of the native RBS suggests a higher expression of Tar, that might raise the sensitivity of the chemotaxis system(Figure 2) (2).
  
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Fig1. (a) Tar expression in UU1250 strain cloned with K1992004 expretion system - strong RBS. (b) Tar expression in UU1250 strain cloned with K1992004 expretion system - Tar native RBS
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[[File:Tar2 swarming.jpeg|600px|thumb|center|Fig1: Tar expression using the native RBS in UU1250 strain, resulting in a halo indicating a functional chemotaxis response. b. Negative control - UU1250 strain w/o the Tar expression plasmid. c. Positive control - ΔZ strain expressing all chemoreceptors.]]
  
  
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The part was successfully sequenced (fig2) ensuring the present of the promoter, RBS, Tar receptor and terminator.
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[[File:Tar1and2.png|600px|thumb|center|Fig2: Comparison between our two new strains UST and UNT: a. UST strain - with the plasmid K1992004. b. UNT strain - with the plasmid K1992005.]]
  
  
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Figure 2
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===Reference===
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1.BI, Shuangyu; LAI, Luhua. Bacterial chemoreceptors and chemoeffectors.Cellular and Molecular Life Sciences, 2015, 72.4: 691-708.<br>‏
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2.SOURJIK, Victor; BERG, Howard C. Functional interactions between receptors in bacterial chemotaxis. Nature, 2004, 428.6981: 437-441.‏
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Fig 2. (a) Sequencing chromatogram of the K1992005 BioBrick- the RBS is marked. (b) The alignment of the sequencing results in compared to the designed sequence - alignment was done using snapgene software.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1992005 SequenceAndFeatures</partinfo>
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Latest revision as of 01:40, 20 October 2016


Tar native RBS expression system (promoter+RBS+coding+terminator)

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 1355
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 184


Introduction

Tar chemoreceptor is one of four native chemoreceptors of E.coli, it is mediate chemotaxis response towards aspartate and away from Ni and Co (1). This device allows the expression of a functionally native Tar chemoreceptor.

Usage and biology

This device is very similar to our BBa_K1992004 device it is only differ by its RBS. Here the expression system is using the native RBS of the Tar receptor as found in the E.coli genome (BBa_K1992000).

Expriments and results

In order to test the device it was transformed to chemoreceptor free strain (UU1250) and tested for chemotaxis ability using [http://2016.igem.org/Team:Technion_Israel/Experiments swarming assay]. As can be seen in figure 1 the cells expressing the Tar chemoreceptor create a halo indicating a functional chemotaxis result.The larger radius of the native RBS suggests a higher expression of Tar, that might raise the sensitivity of the chemotaxis system(Figure 2) (2).

Fig1: Tar expression using the native RBS in UU1250 strain, resulting in a halo indicating a functional chemotaxis response. b. Negative control - UU1250 strain w/o the Tar expression plasmid. c. Positive control - ΔZ strain expressing all chemoreceptors.


Fig2: Comparison between our two new strains UST and UNT: a. UST strain - with the plasmid K1992004. b. UNT strain - with the plasmid K1992005.


Reference

1.BI, Shuangyu; LAI, Luhua. Bacterial chemoreceptors and chemoeffectors.Cellular and Molecular Life Sciences, 2015, 72.4: 691-708.
‏ 2.SOURJIK, Victor; BERG, Howard C. Functional interactions between receptors in bacterial chemotaxis. Nature, 2004, 428.6981: 437-441.‏