Difference between revisions of "Part:BBa K3763021"

 
 
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<partinfo>BBa_K3763021 short</partinfo>
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<!-- Add more about the biology of this part here-->
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==Biology and use==
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Propionicin T1 is the first bacteriocin from propionibacteria. It is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids.<br>
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The mature PctA is reported to be active against many Propionibacterium species, including all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, Propionibacterium jensenii , and in later report, Propionibacterium acnes. <br> <br>
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                <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/6/6e/T--WHU_China--21_Picture1.png" width="40%">
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                <p style="color:Gray; padding:0px 30px 10px;">Figure 1. (A) Growth of the sensitive indicator P. acidipropionici ATCC 4965 with (1,000 BU/ml of culture) or without propionicin T1. Symbols: ◊, without bacteriocin; ■, with bacteriocin. (b) Optical density (620 nm) of growing cultures of P. acidipropionici ATCC 4965 with or without propionicin T1. Symbols: ◊, without bacteriocin; ■, with 100 BU/ml of culture; ▵, with 1,000 BU/ml of culture. [1]</p>
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==Protein sequence and predicted structure==
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                <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/1/1a/T--WHU_China--21_Picture4.png" width="60%">
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                <p style="color:Gray; padding:0px 30px 10px;">Figure 2. The amino acid sequence of PctA.</p>
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The signal peptide is cut off right after Ala-Met-Ala. <br> <br>
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                <p style="color:Gray; padding:0px 30px 10px;">Figure 3. The predicted structure of PctA, predicted by a online website named ColabFold, which is the simplified version of AlphaFold2.</p>
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==Results==
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We’ve successfully expressed precursor PctA in Escherichia coli strain BL21(DE3) using T7 expression system, but it seemed that the pellet of bacteria lysate contained most of the precursor PctA, while the lysate supernatant had a much smaller portion. No mature PctA was found both in the lysate and the culture medium. This indicates that the original Sec signal peptide may not fit in the E.coli chassis. <br>
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        <div class="col-lg" style="margin:auto;text-align:center;">
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                <img style="margin:20px auto 5px auto;" src="https://static.igem.org/mediawiki/parts/2/27/T--WHU_China--21_Picture3.png" width="90%">
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                <p style="color:Gray; padding:0px 30px 10px;">Figure 4. The amino acid sequence of PctA.</p>
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Progress should be made to this part before it can be used to express PctA, cut off the signal peptide and transport it into the environment to kill Propionibacterium acnes. We have tried to change the signal peptide, but we haven’t got results after this modification due to the lack of time.<br>
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Reference <br>
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1.Faye T, Langsrud T, Nes IF, Holo H. Biochemical and genetic characterization of propionicin T1, a new bacteriocin from Propionibacterium thoenii. Appl Environ Microbiol. 2000 Oct;66(10):4230-6. doi: 10.1128/AEM.66.10.4230-4236.2000. PMID: 11010864; PMCID: PMC92290.<br>
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2.Faye T, Holo H, Langsrud T, Nes IF, Brede DA. The unconventional antimicrobial peptides of the classical propionibacteria. Appl Microbiol Biotechnol. 2011 Feb;89(3):549-54. doi: 10.1007/s00253-010-2967-7. Epub 2010 Oct 31. PMID: 21038096.<br>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3763021 SequenceAndFeatures</partinfo>
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Latest revision as of 14:31, 18 October 2021

The coding sequence for bacteriocin propionicin T1

Biology and use

Propionicin T1 is the first bacteriocin from propionibacteria. It is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids.
The mature PctA is reported to be active against many Propionibacterium species, including all strains of Propionibacterium acidipropionici, Propionibacterium thoenii, Propionibacterium jensenii , and in later report, Propionibacterium acnes.

Figure 1. (A) Growth of the sensitive indicator P. acidipropionici ATCC 4965 with (1,000 BU/ml of culture) or without propionicin T1. Symbols: ◊, without bacteriocin; ■, with bacteriocin. (b) Optical density (620 nm) of growing cultures of P. acidipropionici ATCC 4965 with or without propionicin T1. Symbols: ◊, without bacteriocin; ■, with 100 BU/ml of culture; ▵, with 1,000 BU/ml of culture. [1]


Protein sequence and predicted structure

Figure 2. The amino acid sequence of PctA.

The signal peptide is cut off right after Ala-Met-Ala.

Figure 3. The predicted structure of PctA, predicted by a online website named ColabFold, which is the simplified version of AlphaFold2.


Results

We’ve successfully expressed precursor PctA in Escherichia coli strain BL21(DE3) using T7 expression system, but it seemed that the pellet of bacteria lysate contained most of the precursor PctA, while the lysate supernatant had a much smaller portion. No mature PctA was found both in the lysate and the culture medium. This indicates that the original Sec signal peptide may not fit in the E.coli chassis.

Figure 4. The amino acid sequence of PctA.

Progress should be made to this part before it can be used to express PctA, cut off the signal peptide and transport it into the environment to kill Propionibacterium acnes. We have tried to change the signal peptide, but we haven’t got results after this modification due to the lack of time.

Reference
1.Faye T, Langsrud T, Nes IF, Holo H. Biochemical and genetic characterization of propionicin T1, a new bacteriocin from Propionibacterium thoenii. Appl Environ Microbiol. 2000 Oct;66(10):4230-6. doi: 10.1128/AEM.66.10.4230-4236.2000. PMID: 11010864; PMCID: PMC92290.
2.Faye T, Holo H, Langsrud T, Nes IF, Brede DA. The unconventional antimicrobial peptides of the classical propionibacteria. Appl Microbiol Biotechnol. 2011 Feb;89(3):549-54. doi: 10.1007/s00253-010-2967-7. Epub 2010 Oct 31. PMID: 21038096.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 280