Difference between revisions of "Part:BBa K4726001"

 
 
(8 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4726001 short</partinfo>
 
<partinfo>BBa_K4726001 short</partinfo>
 +
===Usage and Biology===
  
jdhdhdhdh
+
This encodes a α-D-galactosamine galactosaminidase from <i>Flavonifractor plautii</i> originating from the human gut microbiota. This enzyme is available on Uniprot under the entry P0DTR5. We added a 6X his-tag at the C-terminus end of the protein.
  
<!-- Add more about the biology of this part here
+
This enzyme works in pair with P0DTR4 (BBa_K4726000) to fully remove the A antigen. It removes the residual α-D-galactosamine that is left after P0DTR4 takes action. P0DTR5 is not active towards A antigen without prior reaction with P0DTR4. The use of both P0DTR4 and P0DTR5 leads to the production of the H antigen, most commonly known as O type blood.
===Usage and Biology===
+
  
<!-- -->
+
The expression of this enzyme led to significant proteotoxicity shown by an impaired growth of <i>Escherichia coli</i> BL21 (DE3). We thus recommended to use Escherichia coli Rosetta (DE3) pLysS which helps stabilizing pET recombinants encoding proteins.
<span class='h3bb'>Sequence and Features</span>
+
<partinfo>BBa_K4726001 SequenceAndFeatures</partinfo>
+
  
 +
===Characterization and results===
 +
The protein was expressed using 0.1 mM IPTG induction in LB broth. After 18 hours of induction at 26 °C, protein were extracted and purified. In well #6, we see overproduction of the aforementioned protein (119.530 KDa).
 +
https://static.igem.wiki/teams/4726/wiki/results/sds-page-purif-wash-elution-igem.png
 +
 +
<b>Figure 1</b>.SDS-PAGE electrophoresis of purification steps.
 +
 +
<!-- -->
 +
<span class='h3bb'><b>Sequence and Features</b></span>
 +
<partinfo>BBa_K4726000 SequenceAndFeatures</partinfo>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
Line 17: Line 24:
 
<partinfo>BBa_K4726001 parameters</partinfo>
 
<partinfo>BBa_K4726001 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
===References===
 +
[1] Rahfeld P, Sim L, Moon H, Constantinescu I, Morgan-Lang C, Hallam SJ, Kizhakkedathu JN, Withers SG. An enzymatic pathway in the human gut microbiome that converts A to universal O type blood. Nat Microbiol. 2019 Sep;4(9):1475-1485. doi: 10.1038/s41564-019-0469-7. Epub 2019 Jun 10. PMID: 31182795.

Latest revision as of 01:20, 10 October 2023


α-D-galactosamine galactosaminidase_Flavonifractor plautii

Usage and Biology

This encodes a α-D-galactosamine galactosaminidase from Flavonifractor plautii originating from the human gut microbiota. This enzyme is available on Uniprot under the entry P0DTR5. We added a 6X his-tag at the C-terminus end of the protein.

This enzyme works in pair with P0DTR4 (BBa_K4726000) to fully remove the A antigen. It removes the residual α-D-galactosamine that is left after P0DTR4 takes action. P0DTR5 is not active towards A antigen without prior reaction with P0DTR4. The use of both P0DTR4 and P0DTR5 leads to the production of the H antigen, most commonly known as O type blood.

The expression of this enzyme led to significant proteotoxicity shown by an impaired growth of Escherichia coli BL21 (DE3). We thus recommended to use Escherichia coli Rosetta (DE3) pLysS which helps stabilizing pET recombinants encoding proteins.

Characterization and results

The protein was expressed using 0.1 mM IPTG induction in LB broth. After 18 hours of induction at 26 °C, protein were extracted and purified. In well #6, we see overproduction of the aforementioned protein (119.530 KDa). sds-page-purif-wash-elution-igem.png

Figure 1.SDS-PAGE electrophoresis of purification steps.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 865
    Illegal EcoRI site found at 1411
    Illegal NgoMIV site found at 1584
    Illegal NgoMIV site found at 1894
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] Rahfeld P, Sim L, Moon H, Constantinescu I, Morgan-Lang C, Hallam SJ, Kizhakkedathu JN, Withers SG. An enzymatic pathway in the human gut microbiome that converts A to universal O type blood. Nat Microbiol. 2019 Sep;4(9):1475-1485. doi: 10.1038/s41564-019-0469-7. Epub 2019 Jun 10. PMID: 31182795.