Difference between revisions of "Part:BBa K4765126"

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This composite part combines [https://parts.igem.org/Part:BBa_K4765016 BBa_K4765016(''H. ex'' mtSSB)],[https://parts.igem.org/Part:BBa_K2306003 BBa_K2306003(SAHS 33020)]and [https://parts.igem.org/Part:BBa_K4765015 BBa_K4765015(AnAFP)] in our ribozyme-assisted polycistronic co-expression system：pRAP.
 
This composite part combines [https://parts.igem.org/Part:BBa_K4765016 BBa_K4765016(''H. ex'' mtSSB)],[https://parts.igem.org/Part:BBa_K2306003 BBa_K2306003(SAHS 33020)]and [https://parts.igem.org/Part:BBa_K4765015 BBa_K4765015(AnAFP)] in our ribozyme-assisted polycistronic co-expression system：pRAP.
  
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AnAFP is an antifreeze protein which endows ''E. coli'' with antifreeze capibility. SAHS 33020 is a heat soluble protein found in the tardigrades which increase ''E. coli'' 's stability against desiccation. ''H. ex'' mtSSB is a type of mitochondrial single-stranded DNA binding protein which improves ''E. coli'' 's resistance to desiccation and UV radiation.
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AnAFP is an antifreeze protein that endows ''E. coli'' with antifreeze capability. SAHS 33020 is a heat-soluble protein found in the tardigrades which increases ''E. coli'' 's stability against desiccation. ''H. ex'' mtSSB is a type of mitochondrial single-stranded DNA binding protein that improves ''E. coli'' 's resistance to desiccation and UV radiation.
  
 
===Characterization===
 
===Characterization===
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We've designed an experimental group with ''E. coli'' expressing ''H. ex'' mtSSB , SAHS 33020 and AnAFP, and used ''E. coli'' transformed with the empty pET28a vector as a control. All proteins are expressed via leakage in ''E. coli'' BL21 DE3. All the groups are incubated overnight. After reaching an OD600 value of 1.0, liquid culture is centrifuged, and the supernatant is removed. Pellets are dried for 6.5 hr in  SpeedVac (Savant SpeedVac SC100) under 4 0 °C .Finally, the pellets are weighed and resuspended in LB medium and dilute 10^5-fold for CFU counting.  
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We've designed an experimental group with ''E. coli'' expressing ''H. ex'' mtSSB, SAHS 33020, and AnAFP, and used ''E. coli'' transformed with the empty pET28a vector as a control. All proteins are expressed via leakage in ''E. coli'' BL21 DE3. All the groups are incubated overnight. After reaching an OD600 value of 1.0, the liquid culture is centrifuged, and the supernatant is removed. Pellets are dried for 6.5 hr in  SpeedVac (Savant SpeedVac SC100) under 4 0 °C. Finally, the pellets are weighed and resuspended in LB medium and dilute 10^5-fold for CFU counting.  
  
 
Through experiments, we observed that when both ''H. ex'' mtSSB and SAHS 33020 are co-expressed, ''E. coli'' exhibits a higher survival rate under desiccation compared to ''E. coli'' expressing these two proteins individually. Furthermore, altering the order of these three proteins in pRAP does not affect the survival rate of ''E. coli'', indicating that pRAP can correctly express these proteins, regardless of their position.
 
Through experiments, we observed that when both ''H. ex'' mtSSB and SAHS 33020 are co-expressed, ''E. coli'' exhibits a higher survival rate under desiccation compared to ''E. coli'' expressing these two proteins individually. Furthermore, altering the order of these three proteins in pRAP does not affect the survival rate of ''E. coli'', indicating that pRAP can correctly express these proteins, regardless of their position.
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/zsl/dry-weight-2-2.png" alt="contributed by Fudan iGEM 2023"></html>
 
| <html><img style="width:400px" src="https://static.igem.wiki/teams/4765/wiki/zsl/dry-weight-2-2.png" alt="contributed by Fudan iGEM 2023"></html>
 
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| '''Figure 3. The dry weight of all the gourps after drying'''
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| '''Figure 3. The dry weight of all the groups after drying'''
 
There is no statistically significant difference in the dry weights among the experimental groups, indicating that the number of ''E. coli'' is consistent between the bacterial tubes.
 
There is no statistically significant difference in the dry weights among the experimental groups, indicating that the number of ''E. coli'' is consistent between the bacterial tubes.
 
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| '''Figure 4. CFU colony count of all the groups after drying'''
 
| '''Figure 4. CFU colony count of all the groups after drying'''
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The ''E. coli'' expressing ''H. ex'' mtSSB, SAHS 33020 and AnAFP together showed higher CFU counts after drying compared to E.coli expresssing ''H. ex'' mtSSB and SAHS33020 individually. Futhermore there's no significant difference between the two ''E. coli''s with rearranged CDS positions. Mean values from three rounds of indepdent experiments are shown. Huge error bars suggest variations between rounds of experiments.
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The ''E. coli'' expressing ''H. ex'' mtSSB, SAHS 33020, and AnAFP together showed higher CFU counts after drying compared to ''E.coli'' expressing ''H. ex'' mtSSB and SAHS33020 individually. Furthermore, there's no significant difference between the two ''E. coli''s with rearranged CDS positions. Mean values from three rounds of independent experiments are shown. Huge error bars suggest variations between rounds of experiments.
 
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Revision as of 15:14, 12 October 2023


ribozyme connected: H. ex mtSSB + SAHS 33020 + AnAFP contributed by Fudan iGEM 2023


Introduction

This composite part combines BBa_K4765016(H. ex mtSSB),BBa_K2306003(SAHS 33020)and BBa_K4765015(AnAFP) in our ribozyme-assisted polycistronic co-expression system：pRAP.

AnAFP is an antifreeze protein that endows E. coli with antifreeze capability. SAHS 33020 is a heat-soluble protein found in the tardigrades which increases E. coli 's stability against desiccation. H. ex mtSSB is a type of mitochondrial single-stranded DNA binding protein that improves E. coli 's resistance to desiccation and UV radiation.

Characterization

Agarose gel electrophoresis

contributed by Fudan iGEM 2023
Figure 1. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures.

From left lane(1) to right lane(3) indicate the successful construction of H. ex mtSSB, H. ex mtSSB + SAHS 33020, and H. ex mtSSB + SAHS 33020 + AnAFP.

Desiccation Survival Assay

We constructed H. ex mtSSB, SAHS 33020, and AnAFP into this part using pRAP. Within BBa_K4765127, we rearranged the positions of these three proteins.

contributed by Fudan iGEM 2023
Figure 2. Workflow of Desiccation Survival Assay

We've designed an experimental group with E. coli expressing H. ex mtSSB, SAHS 33020, and AnAFP, and used E. coli transformed with the empty pET28a vector as a control. All proteins are expressed via leakage in E. coli BL21 DE3. All the groups are incubated overnight. After reaching an OD600 value of 1.0, the liquid culture is centrifuged, and the supernatant is removed. Pellets are dried for 6.5 hr in SpeedVac (Savant SpeedVac SC100) under 4 0 °C. Finally, the pellets are weighed and resuspended in LB medium and dilute 10^5-fold for CFU counting.

Through experiments, we observed that when both H. ex mtSSB and SAHS 33020 are co-expressed, E. coli exhibits a higher survival rate under desiccation compared to E. coli expressing these two proteins individually. Furthermore, altering the order of these three proteins in pRAP does not affect the survival rate of E. coli, indicating that pRAP can correctly express these proteins, regardless of their position.

contributed by Fudan iGEM 2023
Figure 3. The dry weight of all the groups after drying

There is no statistically significant difference in the dry weights among the experimental groups, indicating that the number of E. coli is consistent between the bacterial tubes.

contributed by Fudan iGEM 2023
Figure 4. CFU colony count of all the groups after drying

The E. coli expressing H. ex mtSSB, SAHS 33020, and AnAFP together showed higher CFU counts after drying compared to E.coli expressing H. ex mtSSB and SAHS33020 individually. Furthermore, there's no significant difference between the two E. colis with rearranged CDS positions. Mean values from three rounds of independent experiments are shown. Huge error bars suggest variations between rounds of experiments.

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 247
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 880
    Illegal BsaI.rc site found at 515
    Illegal BsaI.rc site found at 596
    Illegal SapI site found at 409
    Illegal SapI.rc site found at 1804