Difference between revisions of "Part:BBa K1412829"
(→Protocol) |
|||
| Line 47: | Line 47: | ||
===Protocol=== | ===Protocol=== | ||
| − | 1. Transformed <bbpart> | + | 1. Transformed <bbpart>BBa_k1412829</bbpart> into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃. |
2. Inoculate two 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin 50 μg/ml) with single colony from the plate. | 2. Inoculate two 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin 50 μg/ml) with single colony from the plate. | ||
| Line 74: | Line 74: | ||
8. Measure every 30 minutes in the next 4 hrs. | 8. Measure every 30 minutes in the next 4 hrs. | ||
| − | |||
==='''Reference'''=== | ==='''Reference'''=== | ||
Revision as of 05:39, 12 September 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 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 CheZ.
Usage
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 CheZ gene, ending with a TT terminator. Then transfer this gene circuit into E.coli (CheZ knock out), and coat plates, culuture for hours to measure the migration diameter of E.coli.
Relevant parts
Notes
Source 3H:13-P3-3H BBa_R0010 2L:14-P2-2L BBa_B0033 18G:14-P1-18G BBa_K629003 4F:13-P3-4F BBa_B0015
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Protocol
1. Transformed BBa_k1412829 into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃.
2. Inoculate two 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin 50 μg/ml) with single colony from the plate.
3. Cultures were grown in conical flask for 16 hrs at 37℃ with shaking at 200 rpm in the table concentrator.
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.
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.
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.
7. Measure the fluorescence and absorbance:
(1)Fluorescence:
- 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.
- Wavelengths: 501 nm excitation, 514 nm emission, Auto-cutoff: 515 nm.
(2)OD600 (optical density at 600 nm):
- 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.
- Wavelengths: 600 nm absorption.
8. Measure every 30 minutes in the next 4 hrs.