Difference between revisions of "Part:BBa K2918027"

(Characterization of the SSB protein)
(Usage and Biology)
 
(16 intermediate revisions by 3 users not shown)
Line 7: Line 7:
 
The construct is confirmed by sequencing and there are no mutations.
 
The construct is confirmed by sequencing and there are no mutations.
  
===Overview===
+
===Usage and Biology===
 
+
 
The replication of DNA and its conversion into functional proteins are vital processes in all living systems. DNA is copied during the replication process. The bacteriophage Φ29 contains a DNA replication machinery which replicates the linear plasmid by itself. This process is called orthogonal replication and can be beneficially used. The desired gene can be expressed in other hosts without interfering with the genome of its host. Our Sci-Phi 29 tool is based on the Φ29 DNA replication system and its four proteins. The terminal proteins (TP) cap the linear DNA, protect the linear DNA and are the primer for initiation of replication by the Φ29 DNA polymerase (DNAP/p2). DNAP binds to the TP and replicates the DNA. During the replication, single and double stranded DNA is protected by respectively single stranded DNA binding proteins (SSB/p5) and double stranded DNA binding proteins (DSB/p6).
+
The Φ29 replication mechanism involves replication of a protein-primed based replication linear DNA. Protein primed replication, unlike the conventional DNA or RNA primed mechanism, does not depend on specific sequences of DNA/RNA which simplifies the design of replication systems. The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase <html><a href="https://parts.igem.org/Part:BBa_K2918034">(DNAP/p2)</a></html>, terminal protein <html><a href="https://parts.igem.org/Part:BBa_K2918001">(TP/p3)</a></html>, single stranded binding protein <html><a href="https://parts.igem.org/Part:BBa_K2918002"> (SSB/p5)</a></html> and double stranded binding protein <html><a href="https://parts.igem.org/Part:BBa_K2918003">(DSB/p6)</a></html>. The replication process begins by binding of the Φ29 DNA polymerase and terminal protein complex at the origins of replication (<html><a href="https://parts.igem.org/Part:BBa_K2918033">OriL</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K2918061">OriR</a></html>), which flank the protein-primed linear plasmid <html><a href="#Salas1994">(Salas et al., 1994)</a></html>. The double stranded DNA binding proteins aid in the process of replication and bind more intensely at the origins of replication (OriL and OriR), destabilizing the region and facilitating strand displacement. Single stranded binding proteins bind to the displaced DNA strand preventing strand switching of the DNA polymerase and protecting the linear plasmid from host nucleases <html><a href="#Salas1994">(Salas et al., 1994)</a></html>. The replication mechanism is depicted in Figure 1.  If you want to read more about this mechanism, you can take a look at our <html><a target="_blank" href="http://2019.igem.org/Team:TUDelft/Design#orthorep">Design</a></html> page!
 +
 
 +
<div><ul>
 +
<center>
 +
  <li style="display: inline-block;"> [[File:T--TUDelft--replicationpartstest.jpg|thumb|none|550px|<b>Figure 1:</b> Overview of phi29 replication mechanism]] </li>
 +
</center>
 +
    </ul></div>
 +
 
 +
The Φ29 replication system is promising in many ways:
 +
<ul>
 +
<li>The Φ29 DNA polymerase has the highest processivity of all known single subunit DNA polymerases <html><a href="#Blanco1988">(Blanco et al., 1989)</a></html>, and can be used for whole genome amplification. </li>
 +
<li>The Φ29 machinery along with cell-free expression systems can be used to establish the three dogmas of biology <I>in vitro</I>, setting the basis for artificial cell development. </li>
 +
<li>The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide
 +
sequences. </li>
 +
<li>We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many
 +
new engineered protein-DNA complexes.</li>
 +
<li>Orthogonal replication enables not only replication independent from the host, but also the ability to engineer the orthogonal
 +
DNA polymerase’s fidelity without introducing mutations in the cell’s genome which makes <I>in vivo</I> directed evolution a
 +
possibility. </li></ul>
  
 
===Strain Construction===
 
===Strain Construction===
<b>Aim:</b> To clone the promoter in a level 1 MoClo backbone <html><a href="http://www.addgene.org/48003/" target="_blank"> pICH47761</a></html>
+
<b>Aim:</b> To clone the WT T7 promoter, Universal RBS, P5 and T7 terminator in a level 1 MoClo backbone <html><a href="http://www.addgene.org/48003/" target="_blank"> pICH47761</a></html>
 
<br>
 
<br>
 
<b>Procedure:</b> The DNA sequence of the part was cloned with the following Basic parts: <html><a href="https://parts.igem.org/Part:BBa_K2918006" target="_blank"> BBa_K2918006</a>, <a href="https://parts.igem.org/Part:BBa_K2918014" target="_blank"> BBa_K2918014</a>, <a href="https://parts.igem.org/Part:BBa_K2918002" target="_blank"> BBa_K2918002</a> and <a href="https://parts.igem.org/Part:BBa_K2918015" target="_blank"> BBa_K2918015</a></html>. The cloning protocol can be found in the <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank"> protocol</a></html> section of our website!
 
<b>Procedure:</b> The DNA sequence of the part was cloned with the following Basic parts: <html><a href="https://parts.igem.org/Part:BBa_K2918006" target="_blank"> BBa_K2918006</a>, <a href="https://parts.igem.org/Part:BBa_K2918014" target="_blank"> BBa_K2918014</a>, <a href="https://parts.igem.org/Part:BBa_K2918002" target="_blank"> BBa_K2918002</a> and <a href="https://parts.igem.org/Part:BBa_K2918015" target="_blank"> BBa_K2918015</a></html>. The cloning protocol can be found in the <html><a href="http://2019.igem.org/Team:TUDelft/Experiments" target="_blank"> protocol</a></html> section of our website!
Line 18: Line 36:
 
===Characterization of the SSB protein===
 
===Characterization of the SSB protein===
  
For expressing our constructs we used PURE<I>frex</I> 2.0. This is an E.coli based cell-free protein synthesis system and it contains all the elements to make in vitro translation-transcription possible. A 10-μL reaction consists of 5 μL feeding buffer, 0.5 μL enzyme solution, 1 μL ribosome solution, 5 nM DNA and RNAse-free milliQ for filling up the volume. For fluorescent labeling, 0.5 μL of BODIPY-Lys-tRNA<sub>Lys</sub> (FluoroTectTM GreenLys, Promega) was added,  this binds to the translation products at the lysine residues sites.The proteins were identified by an 18% SDS-PAGE gel and mass spectrometry. From the 10-μL reaction, 8 μL was loaded on the SDS-PAGE while the other 2 μL was analysed by the mass spectrometer.
+
For expressing our constructs we used PURE<I>frex</I> 2.0. This is an <I>E. coli</I> based cell-free protein synthesis system and it contains all the elements to make <I>in vitro</I> translation-transcription possible. A 10-μL reaction consists of 5 μL feeding buffer, 0.5 μL enzyme solution, 1 μL ribosome solution, 5 nM DNA and RNAse-free milliQ for filling up the volume. For fluorescent labeling, 0.5 μL of BODIPY-Lys-tRNA<sub>Lys</sub> (FluoroTectTM GreenLys, Promega) was added,  this binds to the translation products at the lysine residues sites.The proteins were identified by an 18% SDS-PAGE gel and mass spectrometry. From the 10 μL reaction, 8 μL was loaded on the SDS-PAGE while the other 2 μL was analysed by mass spectrometry.
  
 
<b>SDS-PAGE</b><br>
 
<b>SDS-PAGE</b><br>
After expressing the SSB protein for 3 hours, the sample was treated with RNAse (RNaseA Solution, Promega) for 30 minutes. To denaturate the protein the sample is also treated with 2x SDS loading buffer with 10 mM dithiotreitol (DTT) for 10 minutes at 90°C. Samples were loaded on a 18% SDS-PAGE (polyacrylamide gel electrophoresis) gel. Visualization was performed on a fluorescence gel imager (Typhoon, Amersham Biosciences) using a 488-nm laser and a band pass emission filter of 520 nm.
+
After expressing the SSB protein for 3 hours, the sample was treated with RNAse (RNaseA Solution, Promega) for 30 minutes. To denature the protein the sample is treated with 2x SDS loading buffer with 10 mM dithiotreitol (DTT) for 10 minutes at 90°C. Samples were loaded on a 18% SDS-PAGE (polyacrylamide gel electrophoresis) gel. Visualization was performed on a fluorescence gel imager (Typhoon, Amersham Biosciences) using a 488 nm laser and a band pass emission filter of 520 nm.
  
 
<div><center><ul>  
 
<div><center><ul>  
<li style="display: inline-block;"> [[File:T--TUDelft--p6666.jpg|thumb|none|850px|<b>Figure 1: SDS-PAGE gels of DSB after protein purification.</b> The gel features the ladder<b>(L)</b>]] </li>
+
<li style="display: inline-block;"> [[File:T--TUDelft--p6666.jpg|thumb|none|850px|<b>Figure 2:</b> SDS-PAGE gels of SSB after cell-free expression. Translation products were analysed by fluorescence imaging of an 18% gel. The bands depicted with an upper red asterisk correspond to the protein of interest with expected molecular weight. In the control line (most right) no specific band can be observed.]] </li>
 
</ul></center></div>
 
</ul></center></div>
  
An SDS-PAGE was carried out for the SSB protein with 3 different promoter strengths: Wild-Type, 0.5 and 0.1. For a control PURE solution without any DNA was used. As can be concluded from the figure, in the sample containing the p5 protein a band indicated by the asterix can be found at the expected molecular weight (13kDa). The band is also absent in the control, indicating that the p5 protein was successfully produced in the PURE system using this construct. The other band that can also be seen in the control could be due to contamination
+
An SDS-PAGE was carried out for the p5 protein with three different promoter strengths (wild-type, medium and weak). To have a negative control, one expression reaction was run in the absence of DNA. In Figure 2, the three samples with different promoter strengths and the control can be seen on the protein gel. The presence of a band (depicted with a red star) at the expected molecular weight (13,3kDa) confirmed the presence of the expressed p5. In the control line with no gene added, a smear background of GreenLys is visible that is distinct from gene-specific bands. The other upper band could be due to contamination in the expression reaction as it is also present in the negative control.
 
+
  
 
<b>Mass Spectrometry </b>
 
<b>Mass Spectrometry </b>
  
Next to the SDS-PAGE, mass spectrometry was used to confirm the identity of the proteins. The mass spectrometer looks for the mass of unique peptide sequences, and their elution time. For p5 these unique peptide sequences are: <I>IFNAQTGGGQSFK</i> and <I>TVAEAASDLIDLVTR</I>. Data was normalized to the presence of the elongation factor EF-TU, which can be found in the same concentration in all PURE system reactions. The raw data and the optimized parameters for the mass spectrometry method can be found [[Media:T--TUDelft--transitionlist.xls.zip|here]].
+
Next to the SDS-PAGE, mass spectrometry was used to confirm the identity of the proteins. The mass spectrometer determines the mass of unique peptide sequences, and their elution time. For p5 these unique peptide sequences are: <I>IFNAQTGGGQSFK</i> and <I>TVAEAASDLIDLVTR</I>. Data was normalized to the presence of the elongation factor EF-TU, which can be found in the same concentration in all PURE system reactions. The raw data and the optimized parameters for the mass spectrometry method can be found [[Media:T--TUDelft--transitionlist.xls.zip|here]] and [[Media:T--TUDelft--RAWDATA.xlsx.zip|here]].
  
  
 
<div><ul>  
 
<div><ul>  
<li style="display: inline-block;"> [[File:T--TUDelft--p5masspec1.png|thumb|none|444px|<b>Figure 2A:</b> Identification in mass spectrometry of one peptide (IFNAQTGGGQSFK) of p6 sample after purification]] </li>
+
<li style="display: inline-block;"> [[File:T--TUDelft--p5masspec1.png|thumb|none|444px|<b>Figure 3A:</b> Identification by mass spectrometry of IFNAQTGGGQSFK peptide in the p5 expressed sample.]] </li>
<li style="display: inline-block;"> [[File:T--TUDelft--p5masspec2.png|thumb|none|444px|<b>Figure 2B:</b> Identification in mass spectrometry of one peptide (TVAEAASDLIDLVTR) of p6 sample after purification]] </li>
+
<li style="display: inline-block;"> [[File:T--TUDelft--p5masspec2.png|thumb|none|444px|<b>Figure 3B:</b> Identification by mass spectrometry of TVAEAASDLIDLVTR in the p5 expressed sample]] </li>
 
</ul></div>
 
</ul></div>
  
The intensity of the mass spectrographs shown in Figure 2 only reflect the <I>occurrence</I> of a given sequence in the sample. These peptide sequences were only present in the samples that were expected. The difference in height can be attributed to the strength of the promoters, less peptides were measured with decreasing strength. For the first peptide <I>IFNAQTGGGQSFK</I>, the intensity of SSB with 0.5 promoter and the 0.1 promoter were 79% and 33% of the intensity of the WT promoter respectively.  For the second peptide <I>TVAEAASDLIDLVTR</I> it is 80% and 37% respectively. In conclusion, the results were positive and the identity of the proteins could be further confirmed by mass spectrometry.
+
The intensity of the mass spectrographs shown in Figure 3 only reflect the <I>occurrence</I> of a given sequence in the sample. These peptide sequences were only present in the samples that were expected. The difference in height can be attributed to the strength of the promoters, less peptides were measured with decreasing strength. For the first peptide <I>IFNAQTGGGQSFK</I>, the intensity of SSB with medium promoter and the weak promoter were 79% and 33% of the intensity of the WT promoter respectively.  For the second peptide <I>TVAEAASDLIDLVTR</I> it is 80% and 37% respectively. In conclusion, the results were positive and the identity of the proteins could be further confirmed by mass spectrometry.
  
 
===Toxicity===
 
===Toxicity===
Line 46: Line 63:
 
Our Sci-Phi 29 tool is based on four components of the Φ29 bacteriophage: DNAP, TP, p5 and p6. However, overexpression of these proteins are toxic for the cell. In order to determine the optimal expression levels of the proteins in live cells, we carried out viability assays in <i>E. coli</i> BL21(DE3) pLysS. The results are shown in the graphs below.
 
Our Sci-Phi 29 tool is based on four components of the Φ29 bacteriophage: DNAP, TP, p5 and p6. However, overexpression of these proteins are toxic for the cell. In order to determine the optimal expression levels of the proteins in live cells, we carried out viability assays in <i>E. coli</i> BL21(DE3) pLysS. The results are shown in the graphs below.
  
<<div><ul>  
+
<div><ul>  
     <li style="display: inline-block;"> [[File:T--TUDelft--noiptgp5.png|thumb|none|444px|<b>Figure 3A:</b> The growth curve of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction]] </li>
+
     <li style="display: inline-block;"> [[File:T--TUDelft--noiptgp5.png|thumb|none|444px|<b>Figure 3A:</b> Normalized maximum growth rate of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction]] </li>
     <li style="display: inline-block;"> [[File:T--TUDelft--1iptgp5.png|thumb|none|444px|<b>Figure 3B:</b> The growth curve of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction]] </li>
+
     <li style="display: inline-block;"> [[File:T--TUDelft--1iptgp5.png|thumb|none|444px|<b>Figure 3B:</b> Normalized maximum growth ratee of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction]] </li>
     <li style="display: inline-block;"> [[File:T--TUDelft--10iptgp5.png|thumb|none|444px|<b>Figure 3C:</b> The growth curve of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction]] </li>
+
     <li> [[File:T--TUDelft--10iptgp5.png|thumb|center|444px|<b>Figure 3C:</b> Normalized maximum growth rate of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction]] </li>
 
     </ul></div>
 
     </ul></div>
  
 +
The growth rate of p5 with a WT promoter is significantly lower than the growth rate of GFP. The trend is also observed upon IPTG induction (Figure 3). However, with a lower promoter strength, we see that the growth is actually better than that of GFP. This could be accounted to the fact that GFP actually also could be toxic as GFP produces H<sub>2</sub>O<sub>2</sub>. It is unclear why GFP toxicity is stronger than that of p5 with lower promoter strength. However, we can state that using a medium promoter the survival of the cells will increase.
 +
 +
If you want to know more, have a look at our <html><a target="_blank" href="http://2019.igem.org/Team:TUDelft/Results#Orthogonality">Results</a></html> page!
 +
 +
===References===
 +
<html>
 +
<ul>
 +
<li>
 +
<a id="Salas1994" href="https://www.pnas.org/content/91/25/12198" target="_blank">
 +
Blanco, L., Lázaro, J. M., De Vega, M., Bonnin, A., & Salas, M. (1994). Terminal protein-primed DNA amplification.<i>Proceedings of the National Academy of Sciences of the United States of America</I>.</a>
 +
</li>
 +
<li>
 +
<a id="Blanco1988" href="https://www.ncbi.nlm.nih.gov/pubmed/2498321" target="_blank">
 +
Blanco, L., Bernads, A., Lharo, J. M., Martins, G., & Garmendia, C. (1989). Highly Efficient DNA Synthesis by the Phage 429 DNA Polymerase.</a>
 +
</li>
 +
 +
</ul>
 +
 +
</html>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 06:24, 14 December 2019


Medium T7 promoter - Universal RBS - Φ29 SSB (p5) - WT T7 terminator 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 68
    Illegal BsaI.rc site found at 94

The construct is confirmed by sequencing and there are no mutations.

Usage and Biology

The Φ29 replication mechanism involves replication of a protein-primed based replication linear DNA. Protein primed replication, unlike the conventional DNA or RNA primed mechanism, does not depend on specific sequences of DNA/RNA which simplifies the design of replication systems. The Φ29 replication can be established by using four simple proteins: Φ29 DNA polymerase (DNAP/p2), terminal protein (TP/p3), single stranded binding protein (SSB/p5) and double stranded binding protein (DSB/p6). The replication process begins by binding of the Φ29 DNA polymerase and terminal protein complex at the origins of replication (OriL and OriR), which flank the protein-primed linear plasmid (Salas et al., 1994). The double stranded DNA binding proteins aid in the process of replication and bind more intensely at the origins of replication (OriL and OriR), destabilizing the region and facilitating strand displacement. Single stranded binding proteins bind to the displaced DNA strand preventing strand switching of the DNA polymerase and protecting the linear plasmid from host nucleases (Salas et al., 1994). The replication mechanism is depicted in Figure 1. If you want to read more about this mechanism, you can take a look at our Design page!

  • Figure 1: Overview of phi29 replication mechanism

The Φ29 replication system is promising in many ways:

  • The Φ29 DNA polymerase has the highest processivity of all known single subunit DNA polymerases (Blanco et al., 1989), and can be used for whole genome amplification.
  • The Φ29 machinery along with cell-free expression systems can be used to establish the three dogmas of biology in vitro, setting the basis for artificial cell development.
  • The existing DNA-protein covalent bonds offer many possibilities to engineer the terminal proteins with functional peptide sequences.
  • We envision that the unique configuration of the double-stranded, protein-capped linear replicon will be a basis for many new engineered protein-DNA complexes.
  • Orthogonal replication enables not only replication independent from the host, but also the ability to engineer the orthogonal DNA polymerase’s fidelity without introducing mutations in the cell’s genome which makes in vivo directed evolution a possibility.

Strain Construction

Aim: To clone the WT T7 promoter, Universal RBS, P5 and T7 terminator in a level 1 MoClo backbone pICH47761
Procedure: The DNA sequence of the part was cloned with the following Basic parts: BBa_K2918006, BBa_K2918014, BBa_K2918002 and BBa_K2918015. The cloning protocol can be found in the protocol section of our website!

Characterization of the SSB protein

For expressing our constructs we used PUREfrex 2.0. This is an E. coli based cell-free protein synthesis system and it contains all the elements to make in vitro translation-transcription possible. A 10-μL reaction consists of 5 μL feeding buffer, 0.5 μL enzyme solution, 1 μL ribosome solution, 5 nM DNA and RNAse-free milliQ for filling up the volume. For fluorescent labeling, 0.5 μL of BODIPY-Lys-tRNALys (FluoroTectTM GreenLys, Promega) was added, this binds to the translation products at the lysine residues sites.The proteins were identified by an 18% SDS-PAGE gel and mass spectrometry. From the 10 μL reaction, 8 μL was loaded on the SDS-PAGE while the other 2 μL was analysed by mass spectrometry.

SDS-PAGE
After expressing the SSB protein for 3 hours, the sample was treated with RNAse (RNaseA Solution, Promega) for 30 minutes. To denature the protein the sample is treated with 2x SDS loading buffer with 10 mM dithiotreitol (DTT) for 10 minutes at 90°C. Samples were loaded on a 18% SDS-PAGE (polyacrylamide gel electrophoresis) gel. Visualization was performed on a fluorescence gel imager (Typhoon, Amersham Biosciences) using a 488 nm laser and a band pass emission filter of 520 nm.

  • Figure 2: SDS-PAGE gels of SSB after cell-free expression. Translation products were analysed by fluorescence imaging of an 18% gel. The bands depicted with an upper red asterisk correspond to the protein of interest with expected molecular weight. In the control line (most right) no specific band can be observed.

An SDS-PAGE was carried out for the p5 protein with three different promoter strengths (wild-type, medium and weak). To have a negative control, one expression reaction was run in the absence of DNA. In Figure 2, the three samples with different promoter strengths and the control can be seen on the protein gel. The presence of a band (depicted with a red star) at the expected molecular weight (13,3kDa) confirmed the presence of the expressed p5. In the control line with no gene added, a smear background of GreenLys is visible that is distinct from gene-specific bands. The other upper band could be due to contamination in the expression reaction as it is also present in the negative control.

Mass Spectrometry

Next to the SDS-PAGE, mass spectrometry was used to confirm the identity of the proteins. The mass spectrometer determines the mass of unique peptide sequences, and their elution time. For p5 these unique peptide sequences are: IFNAQTGGGQSFK and TVAEAASDLIDLVTR. Data was normalized to the presence of the elongation factor EF-TU, which can be found in the same concentration in all PURE system reactions. The raw data and the optimized parameters for the mass spectrometry method can be found here and here.


  • Figure 3A: Identification by mass spectrometry of IFNAQTGGGQSFK peptide in the p5 expressed sample.
  • Figure 3B: Identification by mass spectrometry of TVAEAASDLIDLVTR in the p5 expressed sample

The intensity of the mass spectrographs shown in Figure 3 only reflect the occurrence of a given sequence in the sample. These peptide sequences were only present in the samples that were expected. The difference in height can be attributed to the strength of the promoters, less peptides were measured with decreasing strength. For the first peptide IFNAQTGGGQSFK, the intensity of SSB with medium promoter and the weak promoter were 79% and 33% of the intensity of the WT promoter respectively. For the second peptide TVAEAASDLIDLVTR it is 80% and 37% respectively. In conclusion, the results were positive and the identity of the proteins could be further confirmed by mass spectrometry.

Toxicity

Our Sci-Phi 29 tool is based on four components of the Φ29 bacteriophage: DNAP, TP, p5 and p6. However, overexpression of these proteins are toxic for the cell. In order to determine the optimal expression levels of the proteins in live cells, we carried out viability assays in E. coli BL21(DE3) pLysS. The results are shown in the graphs below.

  • Figure 3A: Normalized maximum growth rate of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction
  • Figure 3B: Normalized maximum growth ratee of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction
  • Figure 3C: Normalized maximum growth rate of phi29 p5 under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction

The growth rate of p5 with a WT promoter is significantly lower than the growth rate of GFP. The trend is also observed upon IPTG induction (Figure 3). However, with a lower promoter strength, we see that the growth is actually better than that of GFP. This could be accounted to the fact that GFP actually also could be toxic as GFP produces H2O2. It is unclear why GFP toxicity is stronger than that of p5 with lower promoter strength. However, we can state that using a medium promoter the survival of the cells will increase.

If you want to know more, have a look at our Results page!

References