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Team:Tokyo Tech/Experiment/RM-lac Hybrid Promoter Assay
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| - | In order to achieve the natural | + | In order to achieve the natural plants' temporal pattern for producing plant hormones in <i>E. coli</i>, we introduced an incoherent feed forward loop, including a new hybrid promoter part ([http://parts.igem.org/Part:BBa_K1139150 BBa_K1139150]). Plants produce their hormones transiently rather than steadily (K. Takei et al., 2001) (Fig. 3-7-1). Moreover, continuous overexpression of hormones is harmful to plants (K. Thiman, 1937). Thus, we thought that it should be important to achieve this transient temporal pattern for producing plant hormones in <i>E. coli</i> too. By applying the incoherent feed forward loop, the transient pulse of gene expression can be generated (S. Basu. S et al., 2003; S. Mangan et al., 2006). |
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[[Image:Titech2013_rmlac_Fig_3-7-1.png|400px|thumb|center|Fig. 3-7-1. Temporal pattern hormones expression in plants]] | [[Image:Titech2013_rmlac_Fig_3-7-1.png|400px|thumb|center|Fig. 3-7-1. Temporal pattern hormones expression in plants]] | ||
Revision as of 10:49, 28 October 2013
RM/lac Hybrid Promoter Assay
Contents |
1. Introduction
In order to achieve the natural plants' temporal pattern for producing plant hormones in E. coli, we introduced an incoherent feed forward loop, including a new hybrid promoter part ([http://parts.igem.org/Part:BBa_K1139150 BBa_K1139150]). Plants produce their hormones transiently rather than steadily (K. Takei et al., 2001) (Fig. 3-7-1). Moreover, continuous overexpression of hormones is harmful to plants (K. Thiman, 1937). Thus, we thought that it should be important to achieve this transient temporal pattern for producing plant hormones in E. coli too. By applying the incoherent feed forward loop, the transient pulse of gene expression can be generated (S. Basu. S et al., 2003; S. Mangan et al., 2006).
Our designed system with an incoherent feed forward loop is shown in Fig. 3-7-2. We newly developed the RM/lac hybrid promoter, which is activated by CI and repressed by LacI (Fig. 3-7-3). We planned to ligate a hormone synthase part downstream of this hybrid promoter. Our mathematical model (Fig. 3-7-4) shows the pulse wave which the temporal pattern for plant hormone production should achieve (details about this model can be found here). While the RM/lac hybrid promoter activation by CI is a single-step reaction, the repression by LacI is a two-step reaction. Thus, the activation of RM/lac hybrid promoter is faster than the repression. This time lag between the activation and the repression is important for generating a temporal pattern of plant hormone production.
 Fig. 3-7-2. Our designed system with an incoherent feed forward loop |
 Fig. 3-7-3. Our newly designed hybrid promoter |
As a first step to achieve this system with an incoherent feed forward loop, we constructed a circuit shown in Fig. 3-7-5 to confirm that our new RM/lac hybrid promoter actually works. We set GFP as a reporter of the RM/lac hybrid promoter and introduced the part into E. coli . We verified that our RM/lac hybrid promoter was activated by CI and repressed by LacI with the following assay.
2. Materials and Methods
Fig. 3-7-6. Constructions for our assay
2-1. Construction
-pSB6A1-Ptet-GFP (N99)…positive control
-pSB6A1-promoterless-GFP (N99)…negative control
-pSB6A1-PRM/lac-GFP (N99)…sample with CI*
-pSB6A1--PRM/lac-GFP (JM2.300)…sample without CI
* This N99 strain expresses CI from its genome constitutively.
2-2. Assay protocol
- Prepare overnight cultures of each cell at 37°C for 12 hours.
- Take 30 microL of the overnight cultures into LB (3 mL) containing antibiotics (Amp 50 microg/mL) and 1 mM of IPTG* (→fresh culture)
*We added IPTG in order to make sure to repress the expression of LacI derived from the E. coli genome.
- After 4 hours of induction, measure the fluorescence intensity with a flow cytometer.
*We added IPTG in order to make sure to repress the expression of LacI derived from the E. coli genome.
3. Results
Fig. 3-7-7 shows the fluorescence intensity detected by flow cytometer. Fig. 3-7-8 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).
 Fig. 3-7-7. Fluorescence intensity detected by flow cytometer |
 Fig. 3-7-8. Comparison of N99 and JM2.300 |
4. Discussion
N99 cells (CI+) showed higher fluorescence intensity than that of JM2.300 cells (CI-). From this result, we assume that our RM/lac hybrid promoter was actually activated by CI. In addition, N99 (IPTG-) showed lower fluorescence than that of N99 (IPTG+). From this result, we can assume that our RM/lac hybrid promoter was repressed by LacI derived from the E. coli genome.
5. References
- Kentaro Takei, Hitoshi Sakakibara, Mitsutaka Taniguchi and Tatsuo Sugiyama. (2001) Nitrogen-Dependent Accumulation of Cytokinins in Root and the Translocation to Leaf: Implication of Cytokinin Species that Induces Gene Expression of Maize Response Regulator. Plant Cell Physiol. 42, 85–93
- Kenneth V. Thiman. (1937) On The Nature of Inhibitions Caused by Auxin. American Journal of Botany. Vol. 24, No. 7, 407-412
- Basu S, Mehreja R, Thiberge S, Chen MT, Weiss R. (2003) Spatiotemporal control of gene expression with pulse-generating networks. Proc. Natl. Acad. Sci. USA, 101, 6355-6360.
- S. Mangan, S. Itzkovitz, A. Zaslaver, U. Alon. (2006) The Incoherent Feed-forward Loop Accelerates the Response-time of the gal System of Escherichia coli . J. Mol. Biol. 356, 1073-1081
