Difference between revisions of "Part:BBa K3370601"

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<br><br><FONT size="5"><i>Introduction</i></FONT><br><br>
 
<br><br><FONT size="5"><i>Introduction</i></FONT><br><br>
 
<br><br><FONT size="4"><i>Gloeobacter</i> rhodopsin introduction</FONT><br><br>
 
<br><br><FONT size="4"><i>Gloeobacter</i> rhodopsin introduction</FONT><br><br>
 
+
 
<p>&emsp;&emsp;GR is a light-driven proton pump that originates from the primitive cyanobacteria, <i>Gloeobacter</i> violaceus. It is a seven helix membrane protein located in the inner membrane. Acting as a light-driven proton pump, GR can transfer protons from the cytoplasmic region to the periplasmic region following light absorption. That is, it establishes the proton motive force to push ATP synthase transforming solar energy into universal energy currency, ATP. The reason that GR has a function with light is its specific chromophore, all-trans-retinal. It changes its conformation when induced by light, resulting in a series of protonated and deprotonated reactions on the several amino acids in GR and causing the transfer of protons.</p>
 
<p>&emsp;&emsp;GR is a light-driven proton pump that originates from the primitive cyanobacteria, <i>Gloeobacter</i> violaceus. It is a seven helix membrane protein located in the inner membrane. Acting as a light-driven proton pump, GR can transfer protons from the cytoplasmic region to the periplasmic region following light absorption. That is, it establishes the proton motive force to push ATP synthase transforming solar energy into universal energy currency, ATP. The reason that GR has a function with light is its specific chromophore, all-trans-retinal. It changes its conformation when induced by light, resulting in a series of protonated and deprotonated reactions on the several amino acids in GR and causing the transfer of protons.</p>
 
   
 
   
<--!{{#tag:html|<img style="width:40%" src="https://2019.igem.org/wiki/images/1/1c/T--NCTU_Formosa--ccdB_modified.png" alt="" />}}!-->
+
{{#tag:html|<img style="width:40%" src="https://2020.igem.org/wiki/images/1/1f/T--NCTU_Formosa--designlinker.png" alt="" />}}
 
+
 
+
 
<br><br><FONT size="4">Modifications of GR for better folding & expression</FONT><br><br>
 
<br><br><FONT size="4">Modifications of GR for better folding & expression</FONT><br><br>
 
+
 
<p>&emsp;&emsp;Harmonized GR is different from the common GR. It's been treated under harmonization, one kind of codon optimization. Since the codon frequency of GR in wild-strain and our host-strain is different, we use harmonization, which is an algorithm, to optimize our sequence of codons but without changing the sequence of amino acids.</p>
 
<p>&emsp;&emsp;Harmonized GR is different from the common GR. It's been treated under harmonization, one kind of codon optimization. Since the codon frequency of GR in wild-strain and our host-strain is different, we use harmonization, which is an algorithm, to optimize our sequence of codons but without changing the sequence of amino acids.</p>
 
+
 
<br><br><FONT size="4">GFP linker vs. Correct Protein Folding</FONT><br><br>
 
<br><br><FONT size="4">GFP linker vs. Correct Protein Folding</FONT><br><br>
 
+
 
<p>&emsp;&emsp;The linker is Gly and Ser rich flexible linker, GSAGSAAGSGEF, which provides performance same as (GGGGS) 4 linker, but it doesn’t have high homologous repeats in DNA coding sequence. Therefore, if GFP expresses well, we can ensure that GR proteins fold robustly and are fully soluble and functional. Furthermore, flexible linker could keep a distance between functional domains, so GFP wouldn’t interfere the function of GR.</p>
 
<p>&emsp;&emsp;The linker is Gly and Ser rich flexible linker, GSAGSAAGSGEF, which provides performance same as (GGGGS) 4 linker, but it doesn’t have high homologous repeats in DNA coding sequence. Therefore, if GFP expresses well, we can ensure that GR proteins fold robustly and are fully soluble and functional. Furthermore, flexible linker could keep a distance between functional domains, so GFP wouldn’t interfere the function of GR.</p>
 
+
 
<br><br><FONT size="5"><i>Results</i></FONT><br><br>
 
<br><br><FONT size="5"><i>Results</i></FONT><br><br>
 
+
 
<FONT size="4">Cloning</FONT><br><br>
 
<FONT size="4">Cloning</FONT><br><br>
 
<p>&emsp;&emsp;We conducted colony PCR to verify that our target gene was correctly cloned into the <i>E. coli</i> Lemo21 (DE3).</p>
 
 
   
 
   
 
+
<p>&emsp;&emsp; We conducted colony PCR to verify that harmonized GR-GFP was correctly cloned into the <i>E. coli</i> Lemo21 (DE3).</p>
<--!{{#tag:html|<img style="width:40%" src="https://2019.igem.org/wiki/images/3/38/T--NCTU_Formosa--ccdB_PCR.png" alt="" />}}!-->
+
 
+
 +
{{#tag:html|<img style="width:40%" src=" https://2020.igem.org/wiki/images/0/07/T--NCTU_Formosa--grwhite.png" alt="" />}}
 +
 
<p class="explanation">
 
<p class="explanation">
         
+
       
 
Figure 1:Colony PCR result of toxin genes after cloning into <i>E. coli</i> Lemo21 (DE3) BBa_K3370001  </p>
 
Figure 1:Colony PCR result of toxin genes after cloning into <i>E. coli</i> Lemo21 (DE3) BBa_K3370001  </p>
 
<br>
 
<br>
 
<p>&emsp;&emsp;Figure 1 was the electrophoresis results of the colony PCR with a marker on the left side and the target gene on the right side. The lengths are labeled beside each band. As a result, we successfully cloned Harmonized GR with GFP linker genes into <i>E. coli</i>.</p>
 
<br>
 
 
 
<FONT size="4">Protein Expression</FONT><br><br>
 
<FONT size="4">Protein Expression</FONT><br><br>
<p>&emsp;&emsp;Express Harmonized GR in pET32a various L-rhamnose concentrations
+
<p>&emsp;&emsp; Expression of harmonized GR-GFP in pET32a with various L-Rhamnose concentrations
From our experimental design, we have tested different L-rhamnose concentration for the best culture environment for GR-expressing system, and we visualized the expression with GFP. We found out that L-rhamnose isn’t needed for GR-expressing <i>E. coli</i> which can also make us know the expression of GR isn’t as hard as we have thought.
+
The proper folding of transmembrane light-induced proton pump(GR) can be visualized
</p>
+
                      by GFP.
 +
It is generally
 +
                      acknowledged that transmembrane
 +
                      proteins are difficult targets for expression, so we chose <i>E. coli</i>, Lemo-21, which features
 +
                      tunable T7 promoter expression system for the expression of GR<sup>[2]</sup>. We found out that GFP expressed
 +
                      best without L-rhamnose inhibition
 +
. Accordingly, <i>Gloeobacter</i> rhodopsin can be easily expressed with proper folding after
 +
                      sequence harmonization, which is good news for GR expression.</p>
 +
 +
 +
{{#tag:html|<img style="width:40%" src=" https://2020.igem.org/wiki/images/b/b0/T--NCTU_Formosa--expresult2.jpg" alt="" />}}
 +
 +
 
   
 
   
 
<--!{{#tag:html|<img style="width:40%" src="https://2019.igem.org/wiki/images/3/38/T--NCTU_Formosa--ccdB_PCR.png" alt="" />}}!-->
 
 
 
 
 
<br><br><FONT size="4">Functional Test</FONT><br><br>
 
<br><br><FONT size="4">Functional Test</FONT><br><br>
 
+
 
<br><FONT size="3">Proton Pump Activity Measurement</FONT><br>
 
<br><FONT size="3">Proton Pump Activity Measurement</FONT><br>
 
+
 
<p>&emsp;&emsp; We measured the proton pumping amount of <i>Gloeobacter</i> rhodopsin by detecting the photocurrent under intervals of light and dark conditions. <i>Gloeobacter</i> rhodopsin expressing <i>E. coli</i> showed a significant increase in photocurrent under light excitation, compared with the vector control, thus proving its proton pumping activity.</p>
 
<p>&emsp;&emsp; We measured the proton pumping amount of <i>Gloeobacter</i> rhodopsin by detecting the photocurrent under intervals of light and dark conditions. <i>Gloeobacter</i> rhodopsin expressing <i>E. coli</i> showed a significant increase in photocurrent under light excitation, compared with the vector control, thus proving its proton pumping activity.</p>
 
+
 
+
<--!{{#tag:html|<img style="width:80%" src="https://2019.igem.org/wiki/images/4/48/T--NCTU_Formosa--ccdB_functional.png" alt="" />}}!-->
+
{{#tag:html|<img style="width:80%" src=" https://2020.igem.org/wiki/images/0/05/T--NCTU_Formosa--photocurrent.jpg" alt="" />}}
 
+
 
<p class="explanation">
 
<p class="explanation">
         
+
          The proton pumping efficiency was determined by the increase in photocurrent at the
Figure2:Proton Pumping Activity of Harmonized GR in pET32a expression system
+
                      duration of illumination. We considered the first illumination to be the genuine representation
From the chart, we can see that when we turned on the light at 420 second, GR pumped proton and changed the pH value outside making the current through the solution increased. When we turned off the light at 540 second, proton flowed back into the cell gradually, pH value increased and the current went through the solution decreased.</p>
+
                      of reflecting the proton pumping activity of GR, so
 
+
                      we took the first duration (420 sec to 540 sec) and analyzed it through proton pumping
 +
                      simulation, and the proton pumping of GR was 0.16 (extracellular, ΔH<sup>+</sup> × 10<sup>-7</sup>/min OD), whereas the
 +
                      value of GR’s proton pumping rate by
 +
                      Pil Kim et al was 0.38
 +
</p>
 +
 
<br><br><FONT size="3">Photototrophic Effect-Growth Measurement</FONT><br><br>
 
<br><br><FONT size="3">Photototrophic Effect-Growth Measurement</FONT><br><br>
 
+
<p>&emsp;&emsp; We measured the growth of GR-expressing E. coli to test the function of our GR expression system.</p>
+
<p>&emsp;&emsp; To further investigate the role of GR-GFP expressed in <i>E. coli</i>, we added sodium
 
+
                  azide to inhibit the respiratory electron transport chain to assess the function of GR-GFP. We
 +
                  hypothesized that GR-GFP’s proton pumping activity could
 +
                  compensate for the loss of function of respiratory electron transport chain due to sodium azide</p>
 +
 
<br><br><FONT size="3"><b>(A)Sodium Azide</b></FONT><br><br>
 
<br><br><FONT size="3"><b>(A)Sodium Azide</b></FONT><br><br>
 
+
 
<p>&emsp;&emsp; We used sodium azide to block the electron transport chain, and assumed the ATP-producing system will be seriously influenced.(More information is in DESIGN) We measured the growth curve to know at light and dark condition, how sodium azide affects GR-expressing E. coli. We found that although it the growth rate of GR-expressing E. coli is also reduced, we discovered that GR really help producing additional ATP for E. coli to use.</p>
 
<p>&emsp;&emsp; We used sodium azide to block the electron transport chain, and assumed the ATP-producing system will be seriously influenced.(More information is in DESIGN) We measured the growth curve to know at light and dark condition, how sodium azide affects GR-expressing E. coli. We found that although it the growth rate of GR-expressing E. coli is also reduced, we discovered that GR really help producing additional ATP for E. coli to use.</p>
 
+
 
+
<--!{{#tag:html|<img style="width:80%" src="https://2019.igem.org/wiki/images/4/48/T--NCTU_Formosa--ccdB_functional.png" alt="" />}}!-->
+
 
+
{{#tag:html|<img style="width:80%" src=" https://2020.igem.org/wiki/images/d/d1/T--NCTU_Formosa--expresult12.jpg" alt="" />}}!
<p class="explanation">
+
         
+
Figure3:Phototrophic growth measurement of GR-expressing E. coli</p>
+
<br>
+
 
+
<--!{{#tag:html|<img style="width:80%" src="https://2019.igem.org/wiki/images/4/48/T--NCTU_Formosa--ccdB_functional.png" alt="" />}}!-->
+
 
+
<p class="explanation">
+
         
+
Figure4:Phototrophic growth measurement of GR-expressing E. coli under sodium azide(0.001%).</p>
+
<br>
+
 
+
<--!{{#tag:html|<img style="width:80%" src="https://2019.igem.org/wiki/images/4/48/T--NCTU_Formosa--ccdB_functional.png" alt="" />}}!-->
+
 
+
 
<p class="explanation">
 
<p class="explanation">
         
+
       
Figure5:Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition.</p>
+
Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition at 20th hour(*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001).</p>
 
<br>
 
<br>
 
+
<--!{{#tag:html|<img style="width:80%" src="https://2019.igem.org/wiki/images/4/48/T--NCTU_Formosa--ccdB_functional.png" alt="" />}}!-->
+
 
+
<p class="explanation">
+
         
+
Figure6:Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition at 20th hour(*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001).</p>
+
<br>
+
 
+
 
<br><FONT size="3"><b>(B)Glucose Consumption</b></FONT><br><br>
 
<br><FONT size="3"><b>(B)Glucose Consumption</b></FONT><br><br>
 
+
<p>&emsp;&emsp;  
+
<p>&emsp;&emsp;
We used low glucose addition M9 minimal medium to express GR and we wanted to know how GR-expression <i>E. coli</i> consumes GR to gain additional ATP. We measured the glucose consumption to know at light and dark condition, how much glucose be consumed. We found that GR-expressing <i>E. coli</i> consumed GR faster, and we successfully built a system for determining, analysis and prediction for the growth curve with the glucose concentration we have inputted into our culture condition.  
+
With respect to the phototrophic growth pattern observed, faster growth of GR-expressing
Glucose Consumption of <i>E. coli</i> WT & GR-expressing <i>E. coli</i>
+
                  <i>E. coli</i> not only relies on the proton gradient, additional ATP, it produces, but also on its
Glucose concentrations are precisely measured in our experiments. We found out that in both light and dark conditions, GR-expressing <i>E. coli</i> consumes glucose much faster than E. coli without GR-expressing system. Therefore, we knew that faster growth of GR-expressing >i>E. coli</i> results from additional ATP produced by GR and faster glucose consumption..</p>
+
                  carbon sources, mass increase, for growth. We
 
+
                  were next interested in finding the consumption rate of glucose in GR-expressing <i>E. coli</i>.
 
+
                  Basically, we expected the higher consumption rate of glucose with additional ATP produced by GR. We
<--!{{#tag:html|<img style="width:80%" src="https://2019.igem.org/wiki/images/4/48/T--NCTU_Formosa--ccdB_functional.png" alt="" />}}!-->
+
                  used M9 medium with glucose (0.4%,
 
+
                  22.2mM), and use DNS reagent to determine the glucose concentration.</p>
 +
 +
 +
{{#tag:html|<img style="width:80%" src=" https://2020.igem.org/wiki/images/4/4b/T--NCTU_Formosa--model-dataglu.png" alt="" />}}
 +
 
<p class="explanation">
 
<p class="explanation">
         
+
       
Figure7: Glucose Consumption of GR-expressing E. coli
+
We found that GR-expressing <i>E. coli</i> consumed GR faster, as it exhausted glucose
From Figure 7, we can see that E. coli with GR used up 22.2mM glucose in 12 hours, and E. coli without GR used up in 14 hours. We found out that GR-expressing <i>E. coli</i> consumes glucose much faster than E. coli without GR-expressing system.</p>
+
                  in 12 hours, while the vector control one (pET32a, Lemo21) took 14 hours for glucose
 
+
                  depletion(Fig.15). The maximum glucose uptake rate(Q<sub>Max</sub>)
 +
                  of GR-expressing <i> E. coli</i> Lemo21 is 11.28(Mm/O.D.600·h) whereas that of vector control one is
 +
                  9.47(Mm/O.D.600·h). Also, we successfully built a system for the prediction for the growth curve
 +
                  with glucose concentration, we have
 +
                  integrated it into our culture condition optimization model</p>
 +
 
<br><br><FONT size="4">Protein Expression Enhancement</FONT><br><br>
 
<br><br><FONT size="4">Protein Expression Enhancement</FONT><br><br>
 
<p>&emsp;&emsp; RFP Expression in GR-expressing Lemo21
 
We expressed RFP in the GR expression system to know how GR-expressing system helps the protein expression.</p>
 
 
<br><FONT size="3"><b>Co-transform BBa_J04450 in psB1K3 & Harmonized GR with GFP linker in pET32a</b></FONT><br><br>
 
 
<p>&emsp;&emsp; We conducted colony PCR to verify that BBa_J04450 and Harmonized GR were both correctly cloned into the E. coli Lemo21 (DE3).</p>
 
 
<--!{{#tag:html|<img style="width:80%" src="https://2019.igem.org/wiki/images/4/48/T--NCTU_Formosa--ccdB_functional.png" alt="" />}}!-->
 
 
<p class="explanation">
 
         
 
Figure8:Colony PCR of co-transform of BBa_J04450(1382 b.p.) &
 
Harmonized GR(2680 b.p.) into E. coli Lemo21
 
</p>
 
<br>
 
 
 
<br><FONT size="3"><b>RFP Expression in GR-expression Lemo21</b></FONT><br>
 
<br><FONT size="3"><b>RFP Expression in GR-expression Lemo21</b></FONT><br>
 
+
<p>&emsp;&emsp; We conducted colony PCR to verify that BBa_J04450 and Harmonized GR were both correctly cloned into the E. coli Lemo21 (DE3).</p>
+
<p>&emsp;&emsp; We cultivated both the GR-expressing <i>E. coli</i> and vector control ones in LB
 
+
                      with IPTG induction in LB broth for incubation. We measured the end point of the final samples
<--!{{#tag:html|<img style="width:80%" src="https://2019.igem.org/wiki/images/4/48/T--NCTU_Formosa--ccdB_functional.png" alt="" />}}!-->
+
                      and compared their RFP fluorescent intensity.</p>
 
+
 +
{{#tag:html|<img style="width:80%" src=" https://2020.igem.org/wiki/images/a/a1/T--NCTU_Formosa--expresult21.jpg" alt="" />}}
 +
 
<p class="explanation">
 
<p class="explanation">
         
+
       
Figure19: RFP expression in GR-expressing E. coli (*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001)
+
RFP expression in GR-expressing E. coli (*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001)
There is 27% increased protein expression when we incorporate target proteins into GR-expressing <i>E. coli</i>
+
GR-expressing <i>E. coli</i> shows stronger fluorescence intensity than the vector
 
+
                      control ones.
 
</p>
 
</p>
 
<br>
 
<br>
 
+
 
+
 +
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 
+
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K3370601 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K3370001 SequenceAndFeatures</partinfo>
 
+
 
+
<!-- Uncomment this to enable Functional Parameter display  
+
<!-- Uncomment this to enable Functional Parameter display
 
===Functional Parameters===
 
===Functional Parameters===
<partinfo>BBa_K3370601 parameters</partinfo>
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<partinfo>BBa_K3370001 parameters</partinfo>
 
<!-- -->
 
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Revision as of 11:14, 27 October 2020


T7 promoter + LacO + RBS + Harmonized GR with linker and GFP + 6x His-tag + Terminator

This composite part is regulated by a T7 promoter. A polyhistidine-tag is used for purification. In order to assess the expression of GR, we used a linker sequence in conjunction with GFP.



Introduction



Gloeobacter rhodopsin introduction

  GR is a light-driven proton pump that originates from the primitive cyanobacteria, Gloeobacter violaceus. It is a seven helix membrane protein located in the inner membrane. Acting as a light-driven proton pump, GR can transfer protons from the cytoplasmic region to the periplasmic region following light absorption. That is, it establishes the proton motive force to push ATP synthase transforming solar energy into universal energy currency, ATP. The reason that GR has a function with light is its specific chromophore, all-trans-retinal. It changes its conformation when induced by light, resulting in a series of protonated and deprotonated reactions on the several amino acids in GR and causing the transfer of protons.



Modifications of GR for better folding & expression

  Harmonized GR is different from the common GR. It's been treated under harmonization, one kind of codon optimization. Since the codon frequency of GR in wild-strain and our host-strain is different, we use harmonization, which is an algorithm, to optimize our sequence of codons but without changing the sequence of amino acids.



GFP linker vs. Correct Protein Folding

  The linker is Gly and Ser rich flexible linker, GSAGSAAGSGEF, which provides performance same as (GGGGS) 4 linker, but it doesn’t have high homologous repeats in DNA coding sequence. Therefore, if GFP expresses well, we can ensure that GR proteins fold robustly and are fully soluble and functional. Furthermore, flexible linker could keep a distance between functional domains, so GFP wouldn’t interfere the function of GR.



Results

Cloning

   We conducted colony PCR to verify that harmonized GR-GFP was correctly cloned into the E. coli Lemo21 (DE3).


Figure 1:Colony PCR result of toxin genes after cloning into E. coli Lemo21 (DE3) BBa_K3370001


Protein Expression

   Expression of harmonized GR-GFP in pET32a with various L-Rhamnose concentrations The proper folding of transmembrane light-induced proton pump(GR) can be visualized by GFP. It is generally acknowledged that transmembrane proteins are difficult targets for expression, so we chose E. coli, Lemo-21, which features tunable T7 promoter expression system for the expression of GR[2]. We found out that GFP expressed best without L-rhamnose inhibition . Accordingly, Gloeobacter rhodopsin can be easily expressed with proper folding after sequence harmonization, which is good news for GR expression.





Functional Test


Proton Pump Activity Measurement

   We measured the proton pumping amount of Gloeobacter rhodopsin by detecting the photocurrent under intervals of light and dark conditions. Gloeobacter rhodopsin expressing E. coli showed a significant increase in photocurrent under light excitation, compared with the vector control, thus proving its proton pumping activity.


The proton pumping efficiency was determined by the increase in photocurrent at the duration of illumination. We considered the first illumination to be the genuine representation of reflecting the proton pumping activity of GR, so we took the first duration (420 sec to 540 sec) and analyzed it through proton pumping simulation, and the proton pumping of GR was 0.16 (extracellular, ΔH+ × 10-7/min OD), whereas the value of GR’s proton pumping rate by Pil Kim et al was 0.38



Photototrophic Effect-Growth Measurement

   To further investigate the role of GR-GFP expressed in E. coli, we added sodium azide to inhibit the respiratory electron transport chain to assess the function of GR-GFP. We hypothesized that GR-GFP’s proton pumping activity could compensate for the loss of function of respiratory electron transport chain due to sodium azide



(A)Sodium Azide

   We used sodium azide to block the electron transport chain, and assumed the ATP-producing system will be seriously influenced.(More information is in DESIGN) We measured the growth curve to know at light and dark condition, how sodium azide affects GR-expressing E. coli. We found that although it the growth rate of GR-expressing E. coli is also reduced, we discovered that GR really help producing additional ATP for E. coli to use.


!

Phototrophic growth measurement of GR-expressing E. coli with/without sodium azide addition at 20th hour(*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001).



(B)Glucose Consumption

   With respect to the phototrophic growth pattern observed, faster growth of GR-expressing E. coli not only relies on the proton gradient, additional ATP, it produces, but also on its carbon sources, mass increase, for growth. We were next interested in finding the consumption rate of glucose in GR-expressing E. coli. Basically, we expected the higher consumption rate of glucose with additional ATP produced by GR. We used M9 medium with glucose (0.4%, 22.2mM), and use DNS reagent to determine the glucose concentration.


We found that GR-expressing E. coli consumed GR faster, as it exhausted glucose in 12 hours, while the vector control one (pET32a, Lemo21) took 14 hours for glucose depletion(Fig.15). The maximum glucose uptake rate(QMax) of GR-expressing E. coli Lemo21 is 11.28(Mm/O.D.600·h) whereas that of vector control one is 9.47(Mm/O.D.600·h). Also, we successfully built a system for the prediction for the growth curve with glucose concentration, we have integrated it into our culture condition optimization model



Protein Expression Enhancement


RFP Expression in GR-expression Lemo21

   We cultivated both the GR-expressing E. coli and vector control ones in LB with IPTG induction in LB broth for incubation. We measured the end point of the final samples and compared their RFP fluorescent intensity.

RFP expression in GR-expressing E. coli (*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001) GR-expressing E. coli shows stronger fluorescence intensity than the vector control ones.



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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 609
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1571