Difference between revisions of "Part:BBa K3034007"

 
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<partinfo>BBa_K3034007 short</partinfo>
 
<partinfo>BBa_K3034007 short</partinfo>
  
We improved a reporter device (<html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>) into a surface presentation + reporting system by fusing GFP with INPNC so that the team could make reporter genes through GFP and anchor the target protein to the bacteria outer membrane for more applications.
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<b>We improved the reporter device (<html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>) into a surface display system (BBa_K3034007) </b>(Fig. 1) by fusing GFP with INPNC (<html><a href='https://parts.igem.org/Part:BBa_K523008'>BBa_K523008</a></html>). 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[1]. INP can anchor one or more "passenger proteins" to the outer membrane of ''E.coli'' DH5α. The fixation of exogenous proteins on the cell 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.
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[[File:T-UESTC-China_GFP_o1.png|400px|thumb|center|'''Fig.1.''' Schematic map of <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html> (Fig. 1a) and <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> (Fig. 1b).]]
  
Besides, we added a segment of linker between INPNC and GFP to ensure that two adjacent domains do not sterically interfere with one another.
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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 bacteria[1].
  
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Besides, we added a segment of linker between INPNC and GFP to ensure that two adjacent domains do not sterically interfere with one another. In our experiments, without linker, GFP could not be properly expressed.
  
 
===Usage and Biology===
 
===Usage and Biology===
  
 
====Quantitative detection of fluorescence====
 
====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 <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html> and ''E.coli'' DH5α carrying BBa_K3034007 (Fig.1).
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First, we cultured the bacteria overnight and adjusted them to the same OD600. We ultrasonic broke, centrifuged and respectively resuspend precipitation to measure the fluorescence intensity of GFP in ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html> and ''E.coli'' DH5α carrying BBa_K3034007 (Fig. 2).
  
[[File:T_UESTC_China_Relative_FI.png|700px|thumb|center|'''Fig.1.''' The relative fluorescence intensity of ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html> and ''E.coli'' DH5α carrying BBa_K3034007.  
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[[File:T_UESTC_China_Relative_FI.png|700px|thumb|center|'''Fig.2.''' The relative fluorescence intensity of ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html> and ''E.coli'' DH5α carrying BBa_K3034007.  
 
The relative fluorescence intensity= Fluorescence of precipitation/ (Fluorescence of supernatant+ Fluorescence of precipitation)×100%.]]
 
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 <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>. But, the content of GFP in the broken ''E.coli'' DH5α with BBa_K3034007 was higher than that in the ''E.coli'' DH5α with <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>.
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The results showed that both precipitation and supernatant contained relatively strong GFP. Moreover, the distribution of GFP in ''E.coli'' DH5α carrying BBa_K3034007 was not significantly different from that in ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>. There are some differences with our expectation, after analysis, it may be caused by incomplete ultrasonic broken of bacteria.
  
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 (with <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>). We hypothesized that INPNC was expressed but not highly active.
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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 the ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>. We hypothesized that INPNC was expressed but the efficiency was not so high.
  
 
====Microscopic observation====
 
====Microscopic observation====
Next, ''E.coli'' DH5α with <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html> (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).
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Next, we used fluorescence microscopy to see if the INPNC worked. ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html> (GFP) was rod-shaped and the fluorescence was equably distributed in ''E.coli'' (Fig. 3a). The fluorescence of ''E.coli'' DH5α carrying BBa_K3034007 (INPNC+GFP) was observed to be dotted and dispersed on the surface of ''E.coli'' (Fig. 3b,3c). The results proved that GFP has apparently been anchored to the surface of the ''E.coli'' and INPNC was working.
  
[[File:T_UESTC_China_INPNC.png|700px|thumb|center|'''Fig.2.''' The fluorescence microscopy of ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>(a) and ''E.coli'' DH5α carrying BBa_K3034007 (b、c). ]]
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In addition, we also noticed that ''E.coli'' DH5α carrying BBa_K3034007 (INPNC+GFP) had fluorescence aggregation on one side of the ''E.coli'' surface (Fig. 3c). The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane. Thus, we are more clear that we have achieved our improvement.
 
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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.
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[[File:T_UESTC_China_INPNC.png|700px|thumb|center|'''Fig.3.''' The fluorescence microscopy of ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html> (a) and ''E.coli'' DH5α carrying BBa_K3034007 (b、c). ]]
  
 
====Conclusion====
 
====Conclusion====
 
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#We improved <html><a href='https://parts.igem.org/Part:BBa_J364000'>BBa_J364000</a></html>. GFP is an intracellular reporter gene, and we achieved the expression of GFP on the surface of E.coli.  
 
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#Based on the current results, there is space for further improvement in the efficiency of system expression (The activity of INPNC needs to be improved).
 
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#Other teams who have the need of surface display can insert their target gene into this system. For example, the system can be applied to whole-cell biocatalysts, heavy metal contamination adsorption, antigen and antibody display, etc[1].
====Improvement and application====
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By fusing GFP with INPNC, we can implement the following improvements:
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# We upgraded this part into a surface display and report system, which can anchor GFP to the surface of ''E.coli'' and realize the function enhancement of the original part.
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# While GFP is used to report the expression of other enzymes, the system can also anchor other enzymes together with GFP to the bacterial surface to realize the surface display of certain enzymes and enhance the enzyme activity.
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====References====
 
====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.
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[1] Li mingya, & Lin chenshui. (2016). Ice crystal nuclear protein and its application in bacterial surface display technology. Amino acids and biological resources, 38(2), 7-11.
  
[2] Lee, S. Y., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends in biotechnology, 21(1), 45-52.
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[2] Qiu, Y., Hudait, A., & Molinero, V. (2019). How Size and Aggregation of Ice-Binding Proteins Control Their Ice Nucleation Efficiency. Journal of the American Chemical Society, 141(18), 7439-7452.
  
  

Latest revision as of 01:57, 22 October 2019


Surface Display System

We improved the reporter device (BBa_J364000) into a surface display system (BBa_K3034007) (Fig. 1) by fusing GFP with INPNC (BBa_K523008). 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.

Fig.1. Schematic map of BBa_J364000 (Fig. 1a) and BBa_K3034007 (Fig. 1b).

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 bacteria[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. In our experiments, without linker, GFP could not be properly expressed.

Usage and Biology

Quantitative detection of fluorescence

First, we cultured the bacteria overnight and adjusted them to the same OD600. We ultrasonic broke, centrifuged and respectively resuspend precipitation to measure the fluorescence intensity of GFP in E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007 (Fig. 2).

Fig.2. 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. Moreover, the distribution of GFP in E.coli DH5α carrying BBa_K3034007 was not significantly different from that in E.coli DH5α carrying BBa_J364000. There are some differences with our expectation, after analysis, it may be caused by incomplete ultrasonic broken of bacteria.

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 the E.coli DH5α carrying BBa_J364000. We hypothesized that INPNC was expressed but the efficiency was not so high.

Microscopic observation

Next, we used fluorescence microscopy to see if the INPNC worked. E.coli DH5α carrying BBa_J364000 (GFP) was rod-shaped and the fluorescence was equably distributed in E.coli (Fig. 3a). The fluorescence of E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) was observed to be dotted and dispersed on the surface of E.coli (Fig. 3b,3c). The results proved that GFP has apparently been anchored to the surface of the E.coli and INPNC was working.

In addition, we also noticed that E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) had fluorescence aggregation on one side of the E.coli surface (Fig. 3c). The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane. Thus, we are more clear that we have achieved our improvement.

Fig.3. The fluorescence microscopy of E.coli DH5α carrying BBa_J364000 (a) and E.coli DH5α carrying BBa_K3034007 (b、c).

Conclusion

  1. We improved BBa_J364000. GFP is an intracellular reporter gene, and we achieved the expression of GFP on the surface of E.coli.
  2. Based on the current results, there is space for further improvement in the efficiency of system expression (The activity of INPNC needs to be improved).
  3. Other teams who have the need of surface display can insert their target gene into this system. For example, the system can be applied to whole-cell biocatalysts, heavy metal contamination adsorption, antigen and antibody display, etc[1].

References

[1] Li mingya, & Lin chenshui. (2016). Ice crystal nuclear protein and its application in bacterial surface display technology. Amino acids and biological resources, 38(2), 7-11.

[2] Qiu, Y., Hudait, A., & Molinero, V. (2019). How Size and Aggregation of Ice-Binding Proteins Control Their Ice Nucleation Efficiency. Journal of the American Chemical Society, 141(18), 7439-7452.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 62
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 472
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1686