Difference between revisions of "Part:BBa K4583058"

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	<partinfo>BBa_K4583058 parameters</partinfo>		<partinfo>BBa_K4583058 parameters</partinfo>
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		+	==Reference==
		+	[1] Gu F, Jiang W, Mu Y, et al. Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS Synth Biol. 2020;9(2):209-217. doi:10.1021/acssynbio.9b00290
		+
		+	[2] Talukder AA, Yanai S, Nitta T, Kato A, Yamada M. RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli. FEBS Lett. 1996;386(2-3):177-180. doi:10.1016/0014-5793(96)00426-7
		+
		+	[3] Shong J, Collins CH. Engineering the esaR promoter for tunable quorum sensing- dependent gene expression. ACS Synth Biol. 2013;2(10):568-575. doi:10.1021/sb4000433
		+
		+	[4] Borrero-de Acuña, J.M., et al., A novel programmable lysozyme-based lysis system in Pseudomonas putida for biopolymer production. Sci Rep, 2017. 7(1): p. 4373.

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Latest revision as of 12:55, 12 October 2023

PYU16-RBS(B0034)-E

PYU16-RBS(B0034)-E

Usage

We designed a auto-lysis system based on this part. The auto-lysis system will express at the late stationary phase and peaks at about 40h.

Late Stationary Phase Promoter

When the bacteria enter the stationary phase, the physiological state of the bacteria changes significantly. During this phase, many genes will respond to make timely adjustments. This 4 parts BBa_K4583000 (PYU3), BBa_K4583001 (PYU7), BBa_K4583003 (PYU16), and BBa_K4583004 (PYU92) are the promoters of E. coil. Their most notable feature is that they will express in the late stationary phase. Moreover, they are self-inducible promoters, which means that no additional inducers are needed to be added for expression. Exogenous inducers are expensive and need to be added artificially, whereas self-induced promoters are cost-effective and relatively stable. This part is also very safe because it comes from E. coli MG1655, a commonly engineered bacterium.

• Late stationary phase promoter
• Self-inducible promoter without additional inducers
• Biosafety

Characterization of Late Stationary Phase Promoter PYU16

Our characterization of this part is divided into two main parts.

• First, this promoter was placed upstream of GFP gene, forming a genetic circuit as shown in Fig. 1. This plasmid was transformed into a bacterium containing another plasmid for characterization. Green and red fluorescence were measured at fixed intervals to compare the expression time and intensity of the two.
• Second, this promoter was placed upstream of the BFP gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts.

For plasmid construction methods and other experimental procedures, see the Design page.

Protocols

Our experimental conditions for characterizing this part were as follows:

• E. coli MG1655
• 30^oC, 48h, under vigorous shaking
• Plasmid Backbone: PACYC
• Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) and Molecular Devices SpectraMax i3x.

We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).

Characterization using GFP in 2-plasmids bacteria

In this section we used the PACYC plasmid with BBa_K4583003(PYU16) upstream of GFP gene(Fig. 1). We transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) plasmids respectively (6 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 2

Only one combination showed significant differences in expression time and expression intensity (L31-PesaRp-PYU16).

Characterization using BFP in 3-plasmids bacteria

In this section, we used the PACYC plasmid with PYU7 upstream of the BFP gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with BBa_K4583011(PesaRp) and BBa_K4583012(PesaS) plasmid respectively (4 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 4.

From the characterization results, we can see that there is a significant delay in the expression of this part from the other promoters. PYU16 is expressed at the stationary phase and peaks at the late stationary phase (42h). We found roughly the same results for both characterizations, but with slightly different onset times. This may be related to the instrumentation used. For this characterization, we used a Molecular Devices SpectraMax i3x, which has a much higher precision. In addition, the difference between the 2-plasmids system and the 3-plasmids system may also account for the difference.

Usage

We designed a auto-lysis system based on this part. The auto-lysis system will express at the late stationary phase and peaks at about 40h.

Late Stationary Phase Promoter

When the bacteria enter the stationary phase, the physiological state of the bacteria changes significantly. During this phase, many genes will respond to make timely adjustments. This 4 parts BBa_K4583000 (PYU3), BBa_K4583001 (PYU7), BBa_K4583003 (PYU16), and BBa_K4583004 (PYU92) are the promoters of E. coil. Their most notable feature is that they will express in the late stationary phase. Moreover, they are self-inducible promoters, which means that no additional inducers are needed to be added for expression. Exogenous inducers are expensive and need to be added artificially, whereas self-induced promoters are cost-effective and relatively stable. This part is also very safe because it comes from E. coli MG1655, a commonly engineered bacterium.

• Late stationary phase promoter
• Self-inducible promoter without additional inducers
• Biosafety

Characterization of Late Stationary Phase Promoter PYU3 (an example)

You can found the results of other 3 promoters in their own page Our characterization of this part is divided into two main parts.

• First, this promoter was placed upstream of GFP gene, forming a genetic circuit as shown in Fig. 1. This plasmid was transformed into a bacterium containing another plasmid for characterization. Green and red fluorescence were measured at fixed intervals to compare the expression time and intensity of the two.
• Second, this promoter was placed upstream of the BFP gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts.

For plasmid construction methods and other experimental procedures, see the Design page.

1. Protocols

Our experimental conditions for characterizing this part were as follows:

• E. coli MG1655
• 30^oC, 48h, under vigorous shaking
• Plasmid Backbone: PACYC
• Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) and Molecular Devices SpectraMax i3x.

We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).

2. Characterization using GFP in 2-plasmids bacteria

In this section we used the PACYC plasmid with PYU3 upstream of GFP gene(Fig. 1). We transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) plasmids respectively (6 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 2

As can be seen from the characterization results, with the exception of three combinations (L19-PesaRp-PYU3, L31-PesaRc-PYU3, and L31-PesaRp-PYU3) the expression time of this element is significantly different from that of the other element in terms of time and intensity.

In the case of the combination L19-PesaRwt-PYU3, for example, the expression of the other part peaked at about 12 h, whereas the expression of this part peaked at about 38 h. The expression of the other part peaked at about 12 h, whereas the expression of this part peaked at about 38 h.

3. Characterization using BFP in 3-plasmids bacteria

In this section, we used the PACYC plasmid with PYU3 upstream of the BFP gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) and BBa_K4583012(PesaS) plasmid respectively (4 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 4.

From the characterization results, we can see that there is a significant delay in the expression of this part from the other promoters. PYU3 is expressed at the stationary phase and peaks at the late stationary phase. We found roughly the same results for both characterizations, but with slightly different onset times. This may be related to the instrumentation used.For this characterization, we used a Molecular Devices SpectraMax i3x, which has a much higher precision. In addition, the difference between the 2-plasmids system and the 3-plasmids system may also account for the difference.

Auto-lysis system

We ligated the E gene to the plasmid backbone we constructed by Gibson's method to obtain PACYC-PYU3-E,PACYC-PYU3-E,PACYC-PYU3-E,PACYC-PYU3-E,PACYC-PYU92-E plasmid(Fig. 3).

In order to enhance its lysis effect in L19 and L31 strains, we decided to do so by increasing the RBS of the promoters of PYU3,PYU7,PYU16. We added RBS named B0031,B0032,B0033,B0034 with four different intensity gradients with RBS intensity of 0.01,0.07,0.3,1. A second experiment was performed.

We succeeded in building the system, but failed to successfully validate it. Our speculation is that PYU3, PYU7, PYU16 and PYU92 are not expressed strongly enough. Our next plan is to replace the stronger RBS. Sequence and Features

Reference

[1] Gu F, Jiang W, Mu Y, et al. Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS Synth Biol. 2020;9(2):209-217. doi:10.1021/acssynbio.9b00290

[2] Talukder AA, Yanai S, Nitta T, Kato A, Yamada M. RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli. FEBS Lett. 1996;386(2-3):177-180. doi:10.1016/0014-5793(96)00426-7

[3] Shong J, Collins CH. Engineering the esaR promoter for tunable quorum sensing- dependent gene expression. ACS Synth Biol. 2013;2(10):568-575. doi:10.1021/sb4000433

[4] Borrero-de Acuña, J.M., et al., A novel programmable lysozyme-based lysis system in Pseudomonas putida for biopolymer production. Sci Rep, 2017. 7(1): p. 4373.