Difference between revisions of "Part:BBa K553001"
 
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<partinfo>BBa_K553001 short</partinfo>
 
<partinfo>BBa_K553001 short</partinfo>
  
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This part generates oc8 HLA synthase
 
  
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The generated protein binds oc8 HLA and acts as a positive modulator on PromoterTraI (BBa_K553002)
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The ''Agrobacterium tumefaciens'' TraI synthase generates the OC8 HLA.
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The gene is located on the Ti plasmid together with TraR (BBa_K553000), the other regulatory component of'' A. Tumefaciens'' quorum sensing.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K553000 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K553001 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K553001 parameters</partinfo>
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===Download Datasheet===
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<partinfo>BBa_K553001 parameters</partinfo>
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<html><div class="table_cell"><center><a title="BBa_K553001" target="_blank" href="https://static.igem.org/mediawiki/parts/0/0f/K553001_V.pdf"><img src="https://static.igem.org/mediawiki/parts/2/27/Units-BBa_K553001.jpg"></a></center></div></html>
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==Improvement and Characterization ==
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Group: [http://2017.igem.org/Team:TokyoTech <b>Tokyo Tech 2017</b>]
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Author: Takuma Yasue
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===Summary of Improvement and Characterization===
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We found that the wild type TraI(<partinfo>BBa_K553001</partinfo>)  did not suit for our genetic circuits because enzyme activity was weak. Thus, we constructed the improved TraI(<partinfo>BBa_K2505033</partinfo>) appropriate to our final circuits.
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===Result ===
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[[File:TraImutationResults.jpg|thumb|left|450px| '''Figure 1:''' '''3OC8HSL production of TraI wild type and mutant'''<br style="clear: both" />Sender <i>E. coli</i>(producing TraI) were grown at 37℃ in liquid LB medium with 1μM of SAM. <i>E. coli</i> introduced empty vector was used as Negative Control.]]<br>
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The result of C8 production using the wild type TraI and mutants(Figure 2) is shown in Figure 1.
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The RFU value of the TraI (K34G)-expressing cells was approximately 3-fold higher than that of the TraI-expressing cells. Other mutant didn’t show improvement of 3OC8HSL production. It was caluculated from calibration curve that the wild type TraI produced 28nM of C8 and TraI (K34G) produced 42nM of C8. [[File:T--TokyoTech--TraIimprove20.jpg|thumb|left|450px| '''Figure 2:''' ''Sequence of traI wild type gene and mutant. '''<br style="clear: both" />The mutations were introduced to the pSB1C3-based traI plasmid using the inverse-PCR method, and successful introduction of the mutations were confirmed with Sanger sequencing.]]
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For more information, visit our page: [http://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement TraI_Improvement page].
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<br style="clear: both" />
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===Discussion===
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In the previous study, it was showned that the E34G mutation of LuxI most likely enhances the interactions between the enzyme and the acyl-ACP substrate. Therefore, we thought that K34G mutation of TraI also has the same effect. <br>
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Taken together, we conclude that we could successfuly increase the productivity of C8. Notably, generation and functional identification the mutant TraI, TraI (K34G), meet the medal criteria of ”parts improvement”, because the wild-type traI parts was registered in iGEM parts collection earlier. However, further improvement of C8 production is necessary to transmit the signal from bacteria to mammalian cells. Such improvement is possible through tuning the experimental conditions further.
  
  

Latest revision as of 09:18, 1 November 2017

A. tumefaciens TraI - OC8 HLA synthase


The Agrobacterium tumefaciens TraI synthase generates the OC8 HLA.

The gene is located on the Ti plasmid together with TraR (BBa_K553000), the other regulatory component of A. Tumefaciens quorum sensing.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 385
  • 1000
    COMPATIBLE WITH RFC[1000]


Download Datasheet


Improvement and Characterization

Group: [http://2017.igem.org/Team:TokyoTech Tokyo Tech 2017]

Author: Takuma Yasue

Summary of Improvement and Characterization

We found that the wild type TraI(BBa_K553001) did not suit for our genetic circuits because enzyme activity was weak. Thus, we constructed the improved TraI(BBa_K2505033) appropriate to our final circuits.

Result

Figure 1: 3OC8HSL production of TraI wild type and mutant
Sender E. coli(producing TraI) were grown at 37℃ in liquid LB medium with 1μM of SAM. E. coli introduced empty vector was used as Negative Control.

The result of C8 production using the wild type TraI and mutants(Figure 2) is shown in Figure 1.

The RFU value of the TraI (K34G)-expressing cells was approximately 3-fold higher than that of the TraI-expressing cells. Other mutant didn’t show improvement of 3OC8HSL production. It was caluculated from calibration curve that the wild type TraI produced 28nM of C8 and TraI (K34G) produced 42nM of C8.
Figure 2:' Sequence of traI wild type gene and mutant.
The mutations were introduced to the pSB1C3-based traI plasmid using the inverse-PCR method, and successful introduction of the mutations were confirmed with Sanger sequencing.

For more information, visit our page: [http://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement TraI_Improvement page].


Discussion

In the previous study, it was showned that the E34G mutation of LuxI most likely enhances the interactions between the enzyme and the acyl-ACP substrate. Therefore, we thought that K34G mutation of TraI also has the same effect.
Taken together, we conclude that we could successfuly increase the productivity of C8. Notably, generation and functional identification the mutant TraI, TraI (K34G), meet the medal criteria of ”parts improvement”, because the wild-type traI parts was registered in iGEM parts collection earlier. However, further improvement of C8 production is necessary to transmit the signal from bacteria to mammalian cells. Such improvement is possible through tuning the experimental conditions further.