Difference between revisions of "Part:BBa K1412829"
 
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<partinfo>BBa_K1412829 short</partinfo>
 
<partinfo>BBa_K1412829 short</partinfo>
  
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BBa_K1412829: <i>Plac-RBS(0.01)-CheZ-TT</i>
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This part consists of a <i>CheZ</i> gene which can express CheZ protein deciding <i>E.coli</i> whether tumble or swim straight.In this light, we can characterize the RBS and promoter efficiency by just change different promoters or ribosome binding sites.Then we can characterize the efficiency of RBS and promoter via measuring the migration distance positively associated with the expression strength of <i>CheZ</i>.
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'''BBa_K1412829: Plac-RBS(0.3)-<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 makes <i>E. coli</i> tumble or swim straight. In this light, we can characterize the RBS efficiency by just change different RBS. Then we can characterize the efficiency of RBS via measuring the migration distance positively associated with the expression strength of CheZ.
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[[File:Characterization_process_design.png |700px|thumb|center|<b>Figure 1</b>.The schematic diagram of how we characterize the activity of promoters.]]
  
  
 
==='''Usage'''===
 
==='''Usage'''===
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When we want to characterize the efficiency of RBS, we usually link the RBS between promoter and  GFP, then characterize the RBS by just measure the fluorescence intensity of GFP. In our part, you need just link RBS after a Plac promoter and before a <i>CheZ </i> gene, ending with a TT terminator. Then transfer this gene circuit into E.coli (<i>CheZ</i> knock out), and coat plates, culuture for hours to measure the migration diameter of <i>E.coli</i>.
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When we want to characterize the efficiency of RBS, we usually connect the RBS with GFP, and then measure the fluorescence intensity of GFP. In our part, you only need to connect RBS after a Plac promoter and before  <i>cheZ</i> gene, ending with a double terminator(Plac-RBS(changeable)-<i>cheZ</i>-TT). Then transferred this gene circuit into <i>E. coli</i> CL-1 (<i>cheZ</i> knocked out), and coated plates, cultured on semi-solid medium to measure the migration diameter of <i>E. coli</i>.
  
  
 
==='''Relevant parts'''===
 
==='''Relevant parts'''===
  
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<bbpart>BBa_K1412005</bbpart>
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<bbpart>BBa_K1412000</bbpart>: pLac-RBS(1.0)-<i>cheZ</i>-TT cheZ generator under pLac promoter
  
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<bbpart>BBa_K1412006</bbpart>
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<bbpart>BBa_K1412005</bbpart>: RBS(1.0)-<i>cheZ</i>-TT
  
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<bbpart>BBa_K1412007</bbpart>
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<bbpart>BBa_K1412006</bbpart>: RBS(0.01)-<i>cheZ</i>-TT
  
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<bbpart>BBa_K1412000</bbpart>
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<bbpart>BBa_K1412007</bbpart>: RBS(0.3)-<i>cheZ</i>-TT
  
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<bbpart>BBa_K1412014</bbpart>: pTet-RBS(1.0)-cheZ-TT Characterize the efficiency of promoters with chemotaxis
  
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<bbpart>BBa_K1412614</bbpart>: pBAD-RBS(1.0)-cheZ-TT Characterize the efficiency of promoters with chemotaxis
  
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==='''Notes'''===
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<bbpart>BBa_K1412801</bbpart>: pLac-RBS(0.01)-cheZ-TT Characterize the efficiency of RBS with chemotaxis
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Source
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3H:13-P3-3H BBa_R0010
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2L:14-P2-2L BBa_B0033
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18G:14-P1-18G BBa_K629003
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4F:13-P3-4F BBa_B0015
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1412829 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1412829 SequenceAndFeatures</partinfo>
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===Protocol===
 
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1. Transformed <bbpart>BBa_k1412924</bbpart> into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃.
 
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2. Inoculate two 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin 50 μg/ml) with single colony from the plate.
 
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3. Cultures were grown in conical flask for 16 hrs at 37℃ with shaking at 200 rpm in the table concentrator.
 
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4. Cultures were diluted 1:100 into 20  ml fresh LB medium and grown for 3 hrs at 37℃ with shaking at 200 rpm in the table concentrator.
 
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5. Then the culture was spun down and washed twice with phosphate-buffered saline ([http://en.wikipedia.org/wiki/Phosphate_buffered_saline PBS], pH 7.4) to minimize the background fluorescence from the medium.
 
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6. The washed cells were suspended in [http://en.wikipedia.org/wiki/Phosphate_buffered_saline PBS] and diluted to bring the cells into an appropriate concentration range (2–5 times) before taking fluorimeter measurements.
 
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7. Measure the fluorescence and absorbance:
 
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(1)Fluorescence:
 
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*Device:  [http://www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/spectramax-m-series-multi-mode-microplate-readers SpectraMax+M5 microplate reader], 96-well plates.
 
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*Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.
 
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(2)OD600 (optical density at 600 nm):
 
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*Device: [http://www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/spectramax-m-series-multi-mode-microplate-readers SpectraMax+M5 microplate reader], 96-well plates.
 
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*Wavelengths: 600 nm absorption.
 
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8. Measure every 30 minutes in the next 4 hrs.
 
  
  
 
==='''Reference'''===
 
==='''Reference'''===
  
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<I><B>More information, click here: [http://2014.igem.org/Team:XMU-China# XMU-China]
  
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<!-- Uncomment this to enable Functional Parameter display  
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<!-- Uncomment this to enable Functional Par
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ameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1412829 parameters</partinfo>
 
<partinfo>BBa_K1412829 parameters</partinfo>
 
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Latest revision as of 00:36, 18 October 2014

Characterize efficiency of RBS with chemotaxis

BBa_K1412829: Plac-RBS(0.3)-cheZ-TT

This part consists of [http://en.wikipedia.org/wiki/Chemotaxis cheZ] gene which can express CheZ protein makes E. coli tumble or swim straight. In this light, we can characterize the RBS efficiency by just change different RBS. Then we can characterize the efficiency of RBS via measuring the migration distance positively associated with the expression strength of CheZ.


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


Usage

When we want to characterize the efficiency of RBS, we usually connect the RBS with GFP, and then measure the fluorescence intensity of GFP. In our part, you only need to connect RBS after a Plac promoter and before cheZ gene, ending with a double terminator(Plac-RBS(changeable)-cheZ-TT). Then transferred this gene circuit into E. coli CL-1 (cheZ knocked out), and coated plates, cultured on semi-solid medium to measure the migration diameter of E. coli.


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_K1412014: pTet-RBS(1.0)-cheZ-TT Characterize the efficiency of promoters with chemotaxis

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


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]


Reference

More information, click here: [http://2014.igem.org/Team:XMU-China# XMU-China]