Difference between revisions of "Part:BBa K2922001"

 
Line 6: Line 6:
  
  
−
<!-- Add more about the biology of this part here
+
 
−
===Usage and Biology===
+
===Biology===
 +
BBa_K2922001 is a composite of Endoglucanase A ([https://parts.igem.org/Part:BBa_K118023 BBa_K118023]) with YebF([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003 BBa_K1659003]), a protein reported to be naturally secreted into the extracellular medium by laboratory''E. coli'' strains:
 +
 
 +
 
 +
'''1. Endoglucanase A'''
 +
 
 +
Cellulose is a polymer composed of beta-1,4-linked glucosyl residues.
 +
Cellulases (endoglucanases), cellobiosidases (exoglucanases), and beta-
 +
glucosidases are required by organisms (some fungi, bacteria) that can
 +
consume it. These enzymes are powerful tools for degradation of plant
 +
cell walls by pathogens and other organisms consuming plant biomass.
 +
 
 +
Bacterium''Cellulomonas fimi''uses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.
 +
 
 +
'''2.YebF'''
 +
 
 +
YebF is a 13kDa protein of unknown function that is perhaps the only protein that has been conclusively documented to be secreted into the extracellular medium by a laboratory ''E. coli'' strain. At the N-terminus, YebF has a 2.2 kDa sec-leader sequence which mediates its translocation through the bacterial inner membrane via the Sec pathway, and is cleaved upon translocation into the periplasm to give the 10.8 kDa "mature" form. Export from periplasm into the extracellular space takes places via the Omp pathway, whereby the electropositive dynamic region of YebF electrostatically helps load YebF onto the OmpF/OmpC porins at their electronegative periplasmic face, and after which the disordered N-terminal region of YebF gets threaded through the OmpF lumen. YebF has been used successfully to mediate the secretion of recombinant proteins
 +
<ref>https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003#Biology</ref>.
 +
 
 +
 
 +
 
 +
===Usage===
 +
In order to let yebF help secrete our cellulase out of the ''E. coli'' membrane, we fused the cellulase gene fragment with yebF gene fragment at the N-terminal by Overlap Extension Polymerase Chain Reaction(OE-PCR), and inserted a flexible GS Linker (GGGGS). PCR product was identified by agarose gel electrophoresis (Fig.1)
 +
 
 +
<html>
 +
    <figure>
 +
        <img src="https://2019.igem.org/wiki/images/1/1b/T--XMU-China--0814.png" height="300" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rm">
 +
<strong>Fig.1</strong> Agarose Gel Electrophoresis of yebF-cenA OE-PCR product. Lane M: Marker.
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
 
 +
 
 +
===Characterization===
 +
These parts were insert into the expression vectors with T7 and RBS (BBa_K525998) by restriction sites ''Eco''RI and ''Pst''I. Then transformed the expression vectors into ''E. coli'' DH5α, and the correct construction of this recombinant plasmid was confirmed by chloramphenicol, colony PCR and plasmid sequencing.
 +
 
 +
We transformed the constructed plasmid into ''E. coli'' BL21 (DE3). After confirmed by the same method, the positive clones were cultivated and induced to express by IPTG. The supernatant of culture medium was obtained by centrifugation. And we gain the total protein by ultrasonic crushing. The lysate was then centrifuged and the supernatant was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining. (Fig. 2)
 +
 
 +
<html>
 +
    <figure>
 +
        <img src="https://2019.igem.org/wiki/images/9/90/T--XMU-China--T7-RBS-yebF-cenA%28R%29_SDS-PAGE.png" width="80%" style="float:center">
 +
        <figcaption>
 +
        <p style="font-size:1rm">
 +
<strong>Fig.2</strong> Fig. s1: SDS-PAGE analysis of E. coli BL21 (DE3) by Sliver staining. YebF-cenA: protein of E. coli BL21 (DE3) carrying T7-RBS-yebF-cenA (BBa_K2922007), target bands can be seen in the medium at 57.5 kDa; Control: protein of E. coli BL21 (DE3) carrying T7 and RBS(BBa_K525998).
 +
        </p>
 +
        </figcaption>
 +
    </figure>
 +
</html>
 +
 
 +
 
 +
 
 +
===References===
 +
<references/>
 +
 
 +
 
 +
 
  
 
<!-- -->
 
<!-- -->

Revision as of 14:35, 19 October 2019


Endoglucanase A fused at N-terminal with YebF secretion protein

This part contains the sequence for the protein endoglucanase A with protein YebF fused to its N-terminus by GS Linker. It can achieve the secretion of Endoglucanase A with the function of YebF protein.


Biology

BBa_K2922001 is a composite of Endoglucanase A (BBa_K118023) with YebF(BBa_K1659003), a protein reported to be naturally secreted into the extracellular medium by laboratoryE. coli strains:


1. Endoglucanase A

Cellulose is a polymer composed of beta-1,4-linked glucosyl residues. Cellulases (endoglucanases), cellobiosidases (exoglucanases), and beta- glucosidases are required by organisms (some fungi, bacteria) that can consume it. These enzymes are powerful tools for degradation of plant cell walls by pathogens and other organisms consuming plant biomass.

BacteriumCellulomonas fimiuses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.

2.YebF

YebF is a 13kDa protein of unknown function that is perhaps the only protein that has been conclusively documented to be secreted into the extracellular medium by a laboratory E. coli strain. At the N-terminus, YebF has a 2.2 kDa sec-leader sequence which mediates its translocation through the bacterial inner membrane via the Sec pathway, and is cleaved upon translocation into the periplasm to give the 10.8 kDa "mature" form. Export from periplasm into the extracellular space takes places via the Omp pathway, whereby the electropositive dynamic region of YebF electrostatically helps load YebF onto the OmpF/OmpC porins at their electronegative periplasmic face, and after which the disordered N-terminal region of YebF gets threaded through the OmpF lumen. YebF has been used successfully to mediate the secretion of recombinant proteins [1].


Usage

In order to let yebF help secrete our cellulase out of the E. coli membrane, we fused the cellulase gene fragment with yebF gene fragment at the N-terminal by Overlap Extension Polymerase Chain Reaction(OE-PCR), and inserted a flexible GS Linker (GGGGS). PCR product was identified by agarose gel electrophoresis (Fig.1)

Fig.1 Agarose Gel Electrophoresis of yebF-cenA OE-PCR product. Lane M: Marker.


Characterization

These parts were insert into the expression vectors with T7 and RBS (BBa_K525998) by restriction sites EcoRI and PstI. Then transformed the expression vectors into E. coli DH5α, and the correct construction of this recombinant plasmid was confirmed by chloramphenicol, colony PCR and plasmid sequencing.

We transformed the constructed plasmid into E. coli BL21 (DE3). After confirmed by the same method, the positive clones were cultivated and induced to express by IPTG. The supernatant of culture medium was obtained by centrifugation. And we gain the total protein by ultrasonic crushing. The lysate was then centrifuged and the supernatant was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining. (Fig. 2)

Fig.2 Fig. s1: SDS-PAGE analysis of E. coli BL21 (DE3) by Sliver staining. YebF-cenA: protein of E. coli BL21 (DE3) carrying T7-RBS-yebF-cenA (BBa_K2922007), target bands can be seen in the medium at 57.5 kDa; Control: protein of E. coli BL21 (DE3) carrying T7 and RBS(BBa_K525998).


References

  1. ↑ https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003#Biology



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 412
    Illegal NotI site found at 1556
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1469
    Illegal BamHI site found at 605
    Illegal XhoI site found at 967
    Illegal XhoI site found at 1216
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 747
    Illegal NgoMIV site found at 1672
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 651