Difference between revisions of "Part:BBa K2152003"

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==Protocols==
+
 
 +
==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 <html><a href="https://parts.igem.org/Part:BBa_K4583000">BBa_K4583000 (PYU3)</a>, <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> are the promoters of <i>E. coil</i>. 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.
 +
* <strong>Late stationary phase promoter</strong>
 +
* <strong>Self-inducible promoter without additional inducers</strong>
 +
* <strong>Biosafety</strong>
 +
===Characterization of Late Stationary Phase Promoter PYU3 (an example)===
 +
<strong>You can found the results of other 3 promoters in their own page</strong>
 +
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.
 +
====1. Protocols====
 +
Our experimental conditions for characterizing this part were as follows:
 +
* <em>E. coli</em> MG1655
 +
* 30<sup>o</sup>C, 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 <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
 +
 
 +
<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>
 +
 
 +
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/pyu3p.png"width="700" height="390">
 +
  <figcaption><b>Fig. 2 </b>. Characterization results of PYU3 in the 2-plasmid bacteria</figcaption>
 +
</figure>
 +
</html>
 +
 
 +
====3. 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>
 +
 
 +
==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).
 
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).
 
<html>
 
<html>
 
<figure>
 
<figure>
 
   <img src="https://static.igem.wiki/teams/4583/wiki/e-lysis-2.png"width="540" height="210">
 
   <img src="https://static.igem.wiki/teams/4583/wiki/e-lysis-2.png"width="540" height="210">
−
   <figcaption><b>Fig. 2 </b>. Genetic circuit of  auto-lysis system based on phi X 174 <i>E</i> gene</figcaption>
+
   <figcaption><b>Fig. 2 </b>. Plasmid construction </figcaption>
 
</figure>
 
</figure>
 
</html>
 
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<figure>
 
<figure>
 
   <img src="https://static.igem.wiki/teams/4583/wiki/e-lysis-2.png"width="540" height="210">
 
   <img src="https://static.igem.wiki/teams/4583/wiki/e-lysis-2.png"width="540" height="210">
−
   <figcaption><b>Fig. 3 </b>. Genetic circuit of  auto-lysis system based on phi X 174 <i>E</i> gene</figcaption>
+
   <figcaption><b>Fig. 3 </b>. Adding RBS on the down stream of promoter</figcaption>
 
</figure>
 
</figure>
 
</html>
 
</html>
 +
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.
 +
==Reference==
 +
[1] Barrell, B.G., G.M. Air, and C.A. Hutchison, 3rd, Overlapping genes in bacteriophage phiX174. Nature, 1976. 264(5581): p. 34-41.
 +
 +
[2] Gu, F., et al., Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS Synth Biol, 2020. 9(2): p. 209-217.
 +
 +
[3] Talukder, A.A., et al., RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli. FEBS Lett, 1996. 386(2-3): p. 177-80.
 +
 +
[4] Gao, Y., et al., Inducible cell lysis systems in microbial production of bio-based chemicals. Appl Microbiol Biotechnol, 2013.

Latest revision as of 13:35, 12 October 2023


Bacteriophage Phi X 174 lysis gene E(wild type)

Lysis protein E spanning the inner and outer membrane of the bacterial, leading to low local degradations of peptidoglycan, allow the release of cytoplasmic content.

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]


Contribution

Group: iGEM Team FZU-China 2021

Author: Jiale Hong

Summary: Successfully demonstrated the lysis function of this gene

Enterobacteria phage phiX174 lysis protein E, also known as ϕX174E, induces host cell lysis. Studies show that ϕX174E can inhibit the activity of the host translocase MraY, which catalyzes the synthesis of lipid I, a necessary step for the host cell wall biosynthesis. We inserted the lysis gene ϕX174E into a plasmid and then transformed the plasmid into the host cell where it was expressed. Protein ϕX174E accumulates inside the cell, and after its concentration reaches a certain threshold, it will induce cell lysis.

We synthesized the ϕX174E gene and ligated it to a pET30a backbone and constructed a plasmid pET30a-ϕX174E. The plasmid was colony PCR-verified and sequencing verified.

We then transformed the plasmid with the correct sequence to BL21(DE3). A single colony was inoculated in a 5 mL fresh LB media, and shaken at 37℃ at 250rpm overnight; then 500 uL of the overnight culture was transferred to a 50 mL fresh media for further growth (OD600 was measured every 30 minutes). When the OD600 reached 1.0, the 50ml cell culture was equally divided into two groups. IPTG was added into one of them (the final concentration of IPTG was 100μM) for lysis protein induction, and the other group does not add IPTG (control). It was observed that the OD600 value of the IPTG group decreased continuously while the OD600 value of the control group continued to increase. It can also be observed by naked eyes that the cell culture in the IPTG group became clear after 2.5 hours of induction, while that in the control group was still cloudy, indicating that bacteria in the IPTG group lysed. The OD600 time courses and pictures of the cell cultures before and after IPTG induction are shown in the figures below:

Fig 1. A time-course result of the lysis induced by ϕX174E. Cells were grown to OD600 = 1 and were induced with 100μM IPTG or an equal amount of water. OD600 values of two groups were measured every 30 minutes.

Fig 2. A photo of cell cultures just before adding water or IPTG.

Fig 3. A photo of cell cultures that were allowed to grow for 2.5 hours after adding water (left) or IPTG (right).

Characterization and improvement contribution made by iGEM23_SDU-CHINA

Group: iGEM 2023 SDU-CHINA

Author: Suiru Lu and Chao Tang

Summary: 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.

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.

Fig. 1 . Genetic circuit of auto-lysis system based on phi X 174 E gene

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
  • 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-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

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

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.

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

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).

Fig. 2 . Plasmid construction
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.
Fig. 3 . Adding RBS on the down stream of promoter
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.

Reference

[1] Barrell, B.G., G.M. Air, and C.A. Hutchison, 3rd, Overlapping genes in bacteriophage phiX174. Nature, 1976. 264(5581): p. 34-41.

[2] Gu, F., et al., Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS Synth Biol, 2020. 9(2): p. 209-217.

[3] Talukder, A.A., et al., RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli. FEBS Lett, 1996. 386(2-3): p. 177-80.

[4] Gao, Y., et al., Inducible cell lysis systems in microbial production of bio-based chemicals. Appl Microbiol Biotechnol, 2013.