Composite

Part:BBa_K1412614

Designed by: Ruihua Zhang   Group: iGEM14_XMU-China   (2014-10-01)
Revision as of 19:04, 17 October 2014 by D (Talk | contribs) (→‎Results)

Characterize the efficiency of promoter(BBa_K206000) with chemotaxis

pBAD- 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 pTet 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.

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. However, no published data telsl us about the relative promoter activity of pBAD (BBa_K206000) because L-arabinose can induce pBAD (BBa_K206000). The characterization of the relative activity of pBAD was carried out with 0.02% inducer L-arabinose in culture. And the ratio (pBAD/pLac) is 0.37.


[http://en.wikipedia.org/wiki/Chemotaxis CheZ]

[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].

[2] https://parts.igem.org/Part:BBa_R0010:Experience

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: pTetR-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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference

[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].

[2] https://parts.igem.org/Part:BBa_R0010:Experience


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

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