Difference between revisions of "Part:BBa K4583000"

(→‎Characterization using fluorescent proteins)
(→‎Reference)
 
(60 intermediate revisions by 2 users not shown)
Line 4: Line 4:
 
PYU3 is the promoter of the gene orf-0464, and it comes from Escherichia coli. It will reach its maximum expression at the late stationary phase.
 
PYU3 is the promoter of the gene orf-0464, and it comes from Escherichia coli. It will reach its maximum expression at the late stationary phase.
 
==Usage and Biology==
 
==Usage and Biology==
−
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 part (BBa_K4583000) is the promoter of the gene <em>orf-0464</em>. Its most notable feature is that it will be expressed in the late stationary phase.
+
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 part <html><a href="https://parts.igem.org/Part:BBa_K4583000"> BBa_K4583000(PYU3)</a></html> is the promoter of the gene <em>orf-0464</em>. Its most notable feature is that it will be expressed in the late stationary phase. Moreover, it is a self-inducible promoter, which means that no additional inducers need 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.
 +
* <strong>Late stationary phase promoter</strong>
 +
* <strong>Self-inducible promoter without additional inducers</strong>
 +
* <strong>Biosafety</strong>
 +
There are 3 parts that have similar features characcterized by our team: <html> <a href="https://parts.igem.org/Part:BBa_K4583001"> BBa_K4583001(PYU7)</a> , <a href="https://parts.igem.org/Part:BBa_K4583003"> BBa_K4583003(PYU16)</a>, and <a href="https://parts.igem.org/Part:BBa_K4583004"> BBa_K4583004(PYU92)</a></html>
 +
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4583000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4583000 SequenceAndFeatures</partinfo>
Line 13: Line 18:
  
 
==Characterization==
 
==Characterization==
 +
Our characterization of this part is divided into two main parts.
 +
* First, this promoter was placed upstream of<em> GFP </em>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 <em> BFP </em> 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===
 
===Protocols===
 
Our experimental conditions for characterizing this part were as follows:
 
Our experimental conditions for characterizing this part were as follows:
Line 18: Line 27:
 
* 30<sup>o</sup>C, 48h,  under vigorous shaking
 
* 30<sup>o</sup>C, 48h,  under vigorous shaking
 
* Plasmid Backbone: PACYC
 
* Plasmid Backbone: PACYC
−
* Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.)
+
* 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).
+
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-min). 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 fluorescent proteins===
+
===Characterization using GFP in 2-plasmids bacteria===
 +
In this section we used the PACYC plasmid with PYU3 upstream of <em>GFP</em> gene(Fig. 1).  We transformed it into L19 and L31 with <html><a href="https://parts.igem.org/Part:BBa_K4583009"> BBa_K4583009(PesaRwt)</a>, <a href="https://parts.igem.org/Part:BBa_K4583010"> BBa_K4583010(PesaRc)</a>, <a href="https://parts.igem.org/Part:BBa_K4583011"> BBa_K4583011(PesaRp)</a></html> plasmids respectively (6 combinations in total) and characterized them using 24-well plates.  The characterization results are shown in Fig. 2
  
−
<img src="https://static.igem.wiki/teams/4583/wiki/pyu3l19l31.png" alt="Figure 2: Fluorescence per cell of yeast cells transformed with a plasmid carrying BBa_K801020 in front of an eGFP-gene. The different alcohol concentrations are indicated." style="display: block; margin: 0 auto; width: 800px;">
+
<html>
 +
<figure>
 +
  <img src="https://static.igem.wiki/teams/4583/wiki/pacycpyu3.png"width="410" height="240">
 +
  <figcaption><b>Fig. 1 </b>. Genetic Circuit when characterizing PYU3 using GFP </figcaption>
 +
</figure>
 +
</html>
  
−
===Comparation with another promoter===
+
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.
 +
 
 +
<html>
 +
<figure>
 +
  <img src="https://static.igem.wiki/teams/4583/wiki/pesar-pyu3.png"width="700" height="390">
 +
  <figcaption><b>Fig. 2 </b>. Characterization results of PYU3 in the 2-plasmid bacteria</figcaption>
 +
</figure>
 +
</html>
 +
 
 +
===Characterization using BFP in 3-plasmids bacteria===
 +
In this section, we used the PACYC plasmid with PYU3 upstream of the <em>BFP</em> gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with  <html><a href="https://parts.igem.org/Part:BBa_K4583009"> BBa_K4583009(PesaRwt)</a>, <a href="https://parts.igem.org/Part:BBa_K4583010"> BBa_K4583010(PesaRc)</a>, <a href="https://parts.igem.org/Part:BBa_K4583011"> BBa_K4583011(PesaRp)</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K4583012"> BBa_K4583012(PesaS)</a> plasmid respectively (4 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 4.
 +
<html>
 +
<figure>
 +
  <img src="https://static.igem.wiki/teams/4583/wiki/pacycpyu3bfp.png"width="410" height="240">
 +
  <figcaption><b>Fig. 3 </b>. Genetic Circuit when characterizing PYU3 using BFP </figcaption>
 +
</figure>
 +
</html>
 +
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.
 +
<html>
 +
<figure>
 +
  <img src="https://static.igem.wiki/teams/4583/wiki/pyu33plasmid.png"width="700" height="450">
 +
  <figcaption><b>Fig. 4 </b>. Characterization results of PYU3 in the 3-plasmids bacteria</figcaption>
 +
</figure>
 +
</html>
  
−
===Comparison of expression time with cell density===
 
 
==Reference==
 
==Reference==
 +
[1] Talukder, A A et al. “RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli.” FEBS letters vol. 386,2-3 (1996): 177-80. doi:10.1016/0014-5793(96)00426-7
 +
 +
[2] 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
 +
 +
[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

Latest revision as of 11:30, 12 October 2023

PYU3

PYU3 is the promoter of the gene orf-0464, and it comes from Escherichia coli. It will reach its maximum expression at the late stationary phase.

Usage and Biology

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 part BBa_K4583000(PYU3) is the promoter of the gene orf-0464. Its most notable feature is that it will be expressed in the late stationary phase. Moreover, it is a self-inducible promoter, which means that no additional inducers need 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

There are 3 parts that have similar features characcterized by our team: BBa_K4583001(PYU7) , BBa_K4583003(PYU16), and BBa_K4583004(PYU92)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

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
  • 30oC, 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-min). 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 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

Fig. 1 . Genetic Circuit when characterizing PYU3 using GFP

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.

Fig. 2 . Characterization results of PYU3 in the 2-plasmid bacteria

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.

Fig. 3 . Genetic Circuit when characterizing PYU3 using BFP
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.
Fig. 4 . Characterization results of PYU3 in the 3-plasmids bacteria

Reference

[1] Talukder, A A et al. “RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli.” FEBS letters vol. 386,2-3 (1996): 177-80. doi:10.1016/0014-5793(96)00426-7

[2] 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

[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