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Team:XMU-China/Project Application RBSpromoter
From 2014.igem.org
Characterization the activity of promoter and efficiency of RBS by chemotaxis
The limitation of fluorescence measurement
Traditionally, the activity of promoters and efficiency of RBS are characterized with fluorescence intensity, of which the measurement quite rely on apparatus. Without relative equipment, some iGEM teams can’t do researches such as interlab study. And the precision and accuracy are determined by apparatus too. What’s more, if we need to measure fluorescence intensity in continuous time, it calls for continuous sampling resulting in accumulated error.
Chemotaxis can do more
As we have already proved that the expression strength of CheZ is positively related with motile ability, we develop a new system to characterize the activity of different promoters and efficiency of RBS by chemotaxis. We adopt variable-controlling approach to compare chemotactic diameter shown as the size of the colony between standard parts and unknown parts by measuring the diameter of colony. The only apparatus we need is a ruler.
In order to verify the reliability of the new system. We construct the three devices with different promoters which have been characterized by published papers[1] (Figure 1). Three variable promoters are pLac (BBa_R0010), pBAD (BBa_K206000), pTet (BBa_R0040).
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Figure 1 Devices with different promoters. |
Promoter activity characterization
We stab all three colonies with different promoters on the same semi-solid culture medium with 0.02% L-arabione and 50μg/ml chloramphenicol added in (Figure 2A). After 36 hours culturing, difference of chemotactic diameters between each colonies could be distinguished as Figure 2B.
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Figure 2A Schematic of spotting bacteria. Figure 2B Culturing with 0.02 L-arabione for 48 hours, distinguish difference of chemotaxis diameters between each colonies is shown. |
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Actually, we keep measuring chemotactic diameters of three colonies at different time and set the diameter of the colony with promoter Lac as 1.0. We got the following table (Table 1). The ratio between each colony diameters is 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). Refer to published papers[1], promoter activity between pTetR and pLac has already been measured, and their ratio (pTetR/pLac) is 1.58. So our system is reliable as it could tell the difference between different promoter activities. However, no published data tell us about the relative promoter activity of pBAD (BBa_K206000), while L-arabinose can induce pBAD (BBa_K206000). We make the first characterization data of pBAD activity relative to pLac with 0.02% L-arabinose added in. And the ratio (pBAD/pLac) is 0.37.
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Table 1 The relative activity of different promoters. |
Extensive application
As the results shown above, we draw a conclusion that this new system works well with little errors. We can also standardize this method to characterize most of the promoters. What’s more, it can be used to characterize the RBS efficiency (Figure 3A), terminator efficiency (Figure 3B) and expression strength of target gene etc (Figure 3C).
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Figure 3A Device to characterize the efficiency different RBS. |
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Figure 3B Device to characterize the efficiency different terminator. |
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Figure 3C Device to characterize the expression of target gene. |
Click the following biobricks to read more about our results:
BBa_K1412614
BBa_K1412014
BBa_K1412000
Reference
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.
