Difference between revisions of "Part:BBa K1412014"

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'''BBa_K1412014: pTetR-RBS(1.0)-<i>CheZ</i>-TT'''
 
'''BBa_K1412014: pTetR-RBS(1.0)-<i>CheZ</i>-TT'''
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This part consists of <i>[http://en.wikipedia.org/wiki/Chemotaxis cheZ] </i> gene which can express CheZ protein make <i>E. coli</i> tumbling or swimming straight. In this light, we linked different promoters before <i>cheZ</i> gene. Then we can characterize the efficiency of promoter via measuring the migration distance positively associated with the expression strength of CheZ protein.
  
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This part consists of <i>[http://en.wikipedia.org/wiki/Chemotaxis CheZ]</i> gene which can express CheZ protein make <i>E.coli</i> tumble or swim straight. In this light, we can characterize the efficiency of promoter by replacing different promoters. Then we can characterize the efficiency of promoter via measuring the migration distance positively associated with the expression strength of CheZ.
 
  
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[[File:Characterization_process_design.png |800px|thumb|center|<b>Figure 1</b>. The schematic diagram of how we characterize the activity of promoters.]]
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[[File:Characterization_process_design.png|500px]]
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When we want to characterize the efficiency of promoter, we usually connect the promoter with GFP, then measuring the fluorescence intensity of GFP. In our part, you just need connect RBS(1.0) after a pTetR promoter and before <i>CheZ</i> gene, ending with double terminator(pTetR-RBS(1.0)-<i>CheZ</i>-TT). Then transfer this gene circuit into <i>E.coli</i> (<i>CheZ</i> knocked out), and coat plates, culuture on semi-solid medium to measure the migration diameter of <i>E.coli</i>.
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When we want to characterize the activity of promoter which have not been measured, we usually connect the promoter with GFP, then measuring the fluorescence intensity of GFP. In our project, We linked those three parts: <bbpart>BBa_K1412000</bbpart>, <bbpart>BBa_K1412014</bbpart> and <bbpart>BBa_K1412614]</bbpart>. Then we transferred those gene circuits into <i>E. coli</i> (<i>cheZ</i> knocked out), coated plates, and cultured on semi-solid medium to measure the migration diameter of <i>E. coli</i> parallelly. We verified whether the activity of pLac and pTetR we measured were consistent with which had been measured[1][2] in the beginning. If so, it means that our method is the feasible and we can use this method to characterize the activity of pBAD.
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==='''Relevant parts'''===
 
==='''Relevant parts'''===
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<!-- Add more about the biology of this part here
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==='''Results'''===
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1412014 SequenceAndFeatures</partinfo>
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==='''Protocol'''===
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[[File:Xmu_project_application_RBSpromoter03.png |800px|thumb|center|<b>Fig 2A.</b>Culturing with 0.02 L-arabione for 48 hours, distinguish difference of chemotaxis diameters between each colonies is shown. <b>Fig 2B.</b> The relative activity of different promoters to pLac.]]
  
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===='''Characterization stage'''====
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We set the diameter of the colony with promoter Lac as 1.0, and plot the data in excel, getting the following table (Figure 3).The ratio between each colony diameters was fixed after 36 hours. If we set the fixed ratio as relative promoter activities, from our characterization, promoter TetR (BBa_R0040) activity is 1.86 relative to promoter Lac (BBa_R0010). Referred to published papers, promoter activity between pTetR and pLac had already been measured, and their ratio (pTetR/pLac) is 1.58[http://www.jbioleng.org/content/3/1/4 [1]][https://parts.igem.org/Part:BBa_R0010:Experience [2]]. So our system is reliable as it could tell the difference between different promoter activities.
  
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1. Transfer the part pTetR-RBS(1.0)-<i>CheZ</i>-TT into <i>E.coli</i> (<i>CheZ</i> knock out), and coat plates, culture for hours to measure the migration diameter of <i>E.coli</i>.
 
  
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1412014 SequenceAndFeatures</partinfo>
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<!-- Add more about the biology of this part here
  
 
==='''Reference'''===
 
==='''Reference'''===
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1. Kelly J R, Rubin A J, Davis J H, et al. Measuring the activity of BioBrick promoters using an in vivo reference standard[J]. Journal of biological engineering, 2009, 3(1): 4.
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[1] [http://www.jbioleng.org/content/3/1/4 Kelly J R, Rubin A J, Davis J H, et al. Measuring the activity of BioBrick promoters using an in vivo reference standard[J]. Journal of biological engineering, 2009, 3(1): 4].
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[2] https://parts.igem.org/Part:BBa_R0010:Experience
  
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2. https://parts.igem.org/Part:BBa_R0010:Experience
 
  
  

Latest revision as of 19:36, 17 October 2014

Characterize the efficiency of pTetR (R0040) with chemotaxis

BBa_K1412014: pTetR-RBS(1.0)-CheZ-TT

This part consists of [http://en.wikipedia.org/wiki/Chemotaxis cheZ] gene which can express CheZ protein make E. coli tumbling or swimming straight. In this light, we linked different promoters before cheZ gene. Then we can characterize the efficiency of promoter via measuring the migration distance positively associated with the expression strength of CheZ protein.


Figure 1. The schematic diagram of how we characterize the activity of promoters.


Usage

When we want to characterize the activity of promoter which have not been measured, we usually connect the promoter with GFP, then measuring the fluorescence intensity of GFP. In our project, We linked those three parts: BBa_K1412000, BBa_K1412014 and BBa_K1412614. Then we transferred those gene circuits into E. coli (cheZ knocked out), coated plates, and cultured on semi-solid medium to measure the migration diameter of E. coli parallelly. We verified whether the activity of pLac and pTetR we measured were consistent with which had been measured[1][2] in the beginning. If so, it means that our method is the feasible and we can use this method to characterize the activity of pBAD.

Relevant parts

BBa_K1412000: pLac-RBS(1.0)-CheZ-TT CheZ generator under pLac promoter

BBa_K1412005: RBS(1.0)-CheZ-TT

BBa_K1412006: RBS(0.01)-CheZ-TT

BBa_K1412007: RBS(0.3)-CheZ-TT

BBa_K1412614: pBAD-RBS(1.0)-CheZ-TT Characterize the efficiency of promoters with chemotaxis

BBa_K1412801: pLac-RBS(0.01)-CheZ-TT Characterize the efficiency of RBS with chemotaxis

BBa_K1412829: pLac-RBS(0.3)-CheZ-TT Characterize efficiency of RBS with chemotaxis


Results

Fig 2A.Culturing with 0.02 L-arabione for 48 hours, distinguish difference of chemotaxis diameters between each colonies is shown. Fig 2B. The relative activity of different promoters to pLac.


We set the diameter of the colony with promoter Lac as 1.0, and plot the data in excel, getting the following table (Figure 3).The ratio between each colony diameters was fixed after 36 hours. If we set the fixed ratio as relative promoter activities, from our characterization, promoter TetR (BBa_R0040) activity is 1.86 relative to promoter Lac (BBa_R0010). Referred to published papers, promoter activity between pTetR and pLac had already been measured, and their ratio (pTetR/pLac) is 1.58[http://www.jbioleng.org/content/3/1/4 [1]][2]. So our system is reliable as it could tell the difference between different promoter activities.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]