Difference between revisions of "Team:UESTC-China/Improve"

Revision as of 14:21, 20 October 2019

description

Overview

This year, we improved this reporter device (BBa_J364000) into a surface display system (BBa_K3034007) by fusing GFP with INPNC. The improved system can anchor the downstream protein of INPNC to the surface of bacteria, while the GFP is used as the reporter gene. So, other teams who have the need of surface display can insert their target gene into this system.

Ice nucleation protein (INP) is a secretory outer membrane protein from Pseudomomas syringae, P.flurorescens and several other Gram-negative bacteria. INP can anchor one or more "passenger proteins" to the outer membrane of bacteria. The fixation of exogenous proteins on the bacteria surface through INPNC can not only greatly improve the efficiency of enzymatic reaction, but also avoid the degradation of exogenous proteins by intracellular enzymes of host cells[1].

Besides, we added a segment of linker between inpnc and gfp to ensure that two adjacent domains do not sterically interfere with one another.

Quantitative detection of fluorescence

We first cultured the bacteria overnight and made OD600 uniform. we ultrasonic broken, centrifuged and respectively resuspend precipitation to measure the distribution of GFP in E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007.

Fig.1. The relative fluorescence intensity of E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007. The relative fluorescence intensity= Fluorescence of precipitation/ (Fluorescence of supernatant+ Fluorescence of precipitation)×100%

The results showed that both precipitation and supernatant contained relatively strong GFP after centrifugation.Moreover, the distribution of GFP in E.coli DH5α with BBa_K3034007 was not significantly different from that in E.coli DH5α with BBa_J364000. But, the content of GFP in the broken E.coli DH5α with BBa_K3034007 was higher than that in the E.coli DH5α with BBa_J364000.

Since the E.coli DH5α carrying BBa_K3034007 expressed GFP, this indirectly indicated that INPNC was successfully expressed. However, the content of GFP in the E.coli DH5α precipitate (cell membrane) carrying BBa_K3034007 was not significantly higher than that in the control group (BBa_J364000). We hypothesized that INPNC was expressed but not highly active.

Microscopic observation

Next, E.coli DH5α with BBa_J364000 (GFP) was observed to be rod-shaped and fluorescently filled with E.coli DH5α under a 40-fold microscope.The fluorescence of E.coli DH5α with BBa_K3034007 (INPNC+GFP) was observed to be dotted and dispersed on the surface of E.coli DH5α. The results proved that INPNC was successfully expressed and functioned.

Fig.2. The fluorescence microscopy of E.coli DH5α carrying BBa_J364000(a) and E.coli DH5α carrying BBa_K3034007 (b、c).

In addition, we also noticed that E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) had fluorescence aggregation on one side of the E.coli DH5α surface. The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.

Conclusions

The part we improved was successfully expressed and worked, but INPNC was inefficient.

References

[1] Yang, X., Sun, S., Wang, H., & Hang, H. (2013). Comparison of autotransporter and ice nucleation protein as carrier proteins for antibody display on the cell surface of Escherichia coli. Prog Biochem Biophys, 40, 1209-19.
[2] Lee, S. Y., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends in biotechnology, 21(1), 45-52.

@@ Line 37: / Line 37: @@
 	  <div class="part">
 		 <div class="bigtitle" id="title_1">Overview </div>
-		  This year, we improved this reporter device (<a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>) into a surface display system (BBa_K3034007) by fusing GFP with INPNC. The improved system can anchor the downstream protein of INPNC to the surface of bacteria, while the GFP is used as the reporter gene. So, other teams who have the need of surface display can insert their target gene into this system.
+		  This year, we improved this reporter device (<a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>) into a surface display system (<a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a>) by fusing GFP with INPNC. The improved system can anchor the downstream protein of INPNC to the surface of bacteria, while the GFP is used as the reporter gene. So, other teams who have the need of surface display can insert their target gene into this system.
 	  </div>
@@ Line 50: / Line 50: @@
 		 <div class="bigtitle" id="title_2">Quantitative detection of fluorescence </div>
 		 <div class="mainbody">
-			  We first cultured the bacteria overnight and made OD600 uniform. we ultrasonic broken, centrifuged and  respectively resuspend precipitation to measure the distribution of GFP in <i>E.coli</i> DH5α carrying BBa_J364000 and <i>E.coli</i> DH5α carrying BBa_K3034007.
+			  We first cultured the bacteria overnight and made OD600 uniform. we ultrasonic broken, centrifuged and  respectively resuspend precipitation to measure the distribution of GFP in <i>E.coli</i> DH5α carrying <a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a> and <i>E.coli</i> DH5α carrying <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a>.
            </div>
                   <div class="picture">
                   <img src="https://static.igem.org/mediawiki/2019/e/ee/T--UESTC-China--Improve_1.png" alt="logo" width="70%">
                   </div>
-		 <div class="words">Fig.1. The relative fluorescence intensity of <i>E.coli</i> DH5α carrying BBa_J364000 and <i>E.coli</i> DH5α carrying BBa_K3034007. The relative fluorescence intensity= Fluorescence of precipitation/ (Fluorescence of supernatant+ Fluorescence of precipitation)×100%</div>
+		 <div class="words">Fig.1. The relative fluorescence intensity of <i>E.coli</i> DH5α carrying <a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a> and <i>E.coli</i> DH5α carrying <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a>. The relative fluorescence intensity= Fluorescence of precipitation/ (Fluorescence of supernatant+ Fluorescence of precipitation)×100%</div>
 		 <div class="mainbody">
-			 The results showed that both precipitation and supernatant contained relatively strong GFP after centrifugation.Moreover, the distribution of GFP in <i>E.coli</i> DH5α with BBa_K3034007 was not significantly different from that in <i>E.coli</i> DH5α with BBa_J364000. But, the content of GFP in the broken <i>E.coli</i> DH5α with BBa_K3034007 was higher than that in the <i>E.coli</i> DH5α with BBa_J364000.
+			 The results showed that both precipitation and supernatant contained relatively strong GFP after centrifugation.Moreover, the distribution of GFP in <i>E.coli</i> DH5α with <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a> was not significantly different from that in <i>E.coli</i> DH5α with <a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>. But, the content of GFP in the broken <i>E.coli</i> DH5α with <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a> was higher than that in the <i>E.coli</i> DH5α with <a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>.
 		 </div><br>
 		 <div class="mainbody">
-			 Since the <i>E.coli</i> DH5α carrying BBa_K3034007 expressed GFP, this indirectly indicated that INPNC was successfully expressed. However, the content of GFP in the <i>E.coli</i> DH5α precipitate (cell membrane) carrying BBa_K3034007 was not significantly higher than that in the control group (with BBa_J364000). We hypothesized that INPNC was expressed but not highly active.
+			 Since the <i>E.coli</i> DH5α carrying <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a> expressed GFP, this indirectly indicated that INPNC was successfully expressed. However, the content of GFP in the <i>E.coli</i> DH5α precipitate (cell membrane) carrying <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a> was not significantly higher than that in the control group (<a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>). We hypothesized that INPNC was expressed but not highly active.
 		 </div>
 	 </div>
@@ Line 72: / Line 72: @@
 		<div class="mainbody">
-		Next, <i>E.coli</i> DH5α with BBa_J364000 (GFP) was observed to be rod-shaped and fluorescently filled with <i>E.coli</i> DH5α under a 40-fold microscope.The fluorescence of <i>E.coli</i> DH5α with BBa_K3034007 (INPNC+GFP) was observed to be dotted and dispersed on the surface of <i>E.coli</i> DH5α. The results proved that INPNC was successfully expressed and functioned.
+		Next, <i>E.coli</i> DH5α with <a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a> (GFP) was observed to be rod-shaped and fluorescently filled with <i>E.coli</i> DH5α under a 40-fold microscope.The fluorescence of <i>E.coli</i> DH5α with <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a> (INPNC+GFP) was observed to be dotted and dispersed on the surface of <i>E.coli</i> DH5α. The results proved that INPNC was successfully expressed and functioned.
          </div>
@@ Line 79: / Line 79: @@
 		</div>
 		<div class="words">
-			Fig.2. The fluorescence microscopy of <i>E.coli</i> DH5α carrying BBa_J364000(a) and <i>E.coli</i> DH5α carrying BBa_K3034007 (b、c).
+			Fig.2. The fluorescence microscopy of <i>E.coli</i> DH5α carrying <a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>(a) and <i>E.coli</i> DH5α carrying <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a> (b、c).
 		</div>
 		<div class="mainbody">
-			In addition, we also noticed that <i>E.coli</i> DH5α carrying BBa_K3034007 (INPNC+GFP) had fluorescence aggregation on one side of the <i>E.coli</i> DH5α surface. The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.
+			In addition, we also noticed that <i>E.coli</i> DH5α carrying <a href="http://parts.igem.org/Part:BBa_BBa_K3034007">BBa_K3034007</a> (INPNC+GFP) had fluorescence aggregation on one side of the <i>E.coli</i> DH5α surface. The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.
 		</div>