Difference between revisions of "Part:BBa K1412829"

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BBa_K1412829: <i>Plac-RBS(0.01)-CheZ-TT</i>
 
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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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 or promoter efficiency by just change different promoters or ribosome binding sites(RBS).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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===Protocol===
 
===Protocol===
  
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1. Transformed <bbpart>BBa_k1412829</bbpart> into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃.
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genetic link stage
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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.
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==='''Reference'''===
 
==='''Reference'''===

Revision as of 05:47, 1 October 2014

Characterize efficiency of RBS with chemotaxis

BBa_K1412829: Plac-RBS(0.01)-CheZ-TT

This part consists of a CheZ gene which can express CheZ protein deciding E.coli whether tumble or swim straight.In this light, we can characterize the RBS or promoter efficiency by just change different promoters or ribosome binding sites(RBS).Then we can characterize the efficiency of RBS and promoter via measuring the migration distance positively associated with the expression strength of CheZ.


Usage

When we want to characterize the efficiency of RBS, we usually link the RBS with 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 CheZ gene, ending with a TT terminator(Plac-RBS(changeable)-CheZ-TT). Then transfer this gene circuit into E.coli (CheZ knock out), and coat plates, culuture on semi-solid medium to measure the migration diameter of E.coli.

Relevant parts

BBa_K1412005

BBa_K1412006

BBa_K1412007

BBa_K1412000


Notes

Source 3H:13-P3-3H BBa_R0010