Team:Tokyo Tech/Project/wolf coli/New Series of PompC

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Overview of new OmpC promoter series

We constructed BioBrick parts of the new series of osmoregulative promoters which are derivatives of the wild type OmpC promoter. The new series of promoters are PompC(C), PompC(CB) and PompC(CS1). In order to measure the strength of each promoter, we used GFP as a reporter. We found that expression of GFP in OmpC(CB) and OmpC(CS1) promoters increased in high osmolarity medium comparing with the expression in low osmolarity medium. In contrast, under same conditions, there is no significant difference of GFP expression in OmpC(C) and OmpC(WT) promoters.

• New BioBrick Parts

Figure 1. The induction of new OmpC series in high osmolarity medium

We succeeded in designing 2 news osmoregulative promoters, POmpC(CB) and POmpC(CS1), which can also be utilized in light sensitive system.

Introduction

OmpC promoter, a part of our band detector circuit, plays a crucial role in initiating the transformation of sympathetic coli. into Werewolf coli. in light sensitive system. To accomplish band detector circuit in light sensitive system, varieties of OmpC promoters were designed and characterized in order to find an appropriate strength of the promoter particularly which can be activated by the “full moon light”.

• What is OmpC promoter?

OmpC(WT) promoter (BBa_R0082) is positively regulated by phosphorylated OmpR, a response regulator in EnvZ-OmpR two-component system. The EnvZ-OmpR system of E. coli regulates the transcriptional activity of this promoter in response to change in extracellular osmolarity. The OmpC(WT) promoter consists of 3 distinct phosphorylated OmpR recognition sites which are binding site 1, binding site 2 and binding site 3.

• The new series of OmpC promoters

Three new promoters, PompC(CS1) BBa_395303, PompC(C) BBa_395301, PompC(CB) BBa_395302 were designing based on Maeda S. & Mizuno T. Activation of the Osmoregulated ompC Gene by the OmpR Protein in Escherichia coli. J.Biochem 1991;110,324-27[1]. The second promoter, PompC(CS1), comprises only binding site1 fused to the canonical -35 and -10 region. PompC-C is a composite of binding site 1 and additional 4 base pairs before the canonical -35 and -10 region. Likewise, PompC-CB consists of binding site1 and the additional 10 base pairs before the -35 and -10 consensus sequence.

Material and Method

• Construction of E. coli strain MG1655

Each new BioBrick part, PompC(C)-GFP, PompC(CB)-GFP, PompC(CS1)-GFP and PompC(WT)-GFP on pSB3K3 was introduced into E. coli strain MG1655. A strain containing pLacq-GFP plasmid (BBa_J54202), a constitutive GFP expressive promoter, and promoterless GFP reporter plasmid (BBa_J54103) was used as a positive control and negative control respectively.

• Medium

Medium A, per liter, contained 7 g of nutrient broth, 1 g of yeast extract, 2 g of glycerol, 3.7 g of K2HPO4, and 1.3 g of KH2PO4. [3]

• Report assay

In order to determine the strength of promoter parts in the reporter plasmid, the change of fluorescence intensities of the reporter stains in the presence and the absence of the inducer were measured. Overnight cultures of reporter strains grown at 37 °C in Medium A containing appropriated antibiotics were diluted at least 1:100 in the medium and incubated at 37 °C as fresh cultures. After their OD₅₉₀ reached 0.2, the fresh culture was diluted 1 : 3 into 2 ml of pre-warmed medium A. For high osmolarity conditions, the cultures were diluted with sucrose supplemented medium to the final concentration of 15% (wt/vol). [2]

• Sample preparation for GFP measurement

After 4 hours of high ormolarity induction, 0.2 ml of each culture was moved to 2.0 ml eppendorf tube and then centrifuged for 1 min at 4°C, 9000 rpm. The supernatant was discarded from each tube by pipette. In order to adjust each sample to reach approximately same final OD₅₉₀, appropriate amount of 1×PBS washing solution was added. The cell pellet in each tube was resuspended by vortexing. After all samples for GFP measurement were prepared, 150 μl of each sample was applied to 96-well plate and its fluorescence intensity was measured with fluorometer. The fluorescence intensity was calculated by dividing the measured raw fluorescence intensity by the adjusted OD₅₉₀.

Result

Characterization of new series of OmpC promoters

Figure 1. The induction of new OmpC series in high osmolarity medium

After 4 hours of sucrose induction, transcriptional activity of PompC(CB)-GFP and PompC(CS1)-GFP increased 2.5 folds and 2.3 folds respectively. However, significant amount of leaky expression was found in PompC(CS1)-GFP without induction. In contrast, under the same conditions, we found no significant difference of GFP expression in PompC(C)-GFP and PompC(WT)-GFP.

References

1. Maeda S. & Mizuno T. Activation of the Osmoregulated ompC Gene by the OmpR Protein in Escherichia coli. J.Biochem 1991;110,324-27
2. Batchelor E. & Goulian M. Imaging OmpR localization in Escherichia coli. Molecular Microbiology. 2006;59(6),1767-78
3. Kawaji H. Influence of Molecular Size and Osmolarity of Sugars and Dextrans on the Synthesis of Outer Membrane Proteins 0-8 and 0-9 of Escherichia coli K-12. J.Bacteriology 1979;140(3), 843-47