Difference between revisions of "Part:BBa K2599014"
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===Cloning===
 
===Cloning===
  
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We conbined our toxic gene to pSB1C3 backbone and conducted PCR to check the size of our part. The Leucocyclicin Q sequence length is around 186 b.p. For the composite part, the sequence length should be near at 1230 b.p.
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We conbined our toxic gene to pSB1C3 backbone by the two restriction sites, EcoRI and SpeI, and conducted PCR to check the size of our part. The Leucocyclicin Q sequence length is around 186 b.p. For the composite part, the sequence length should be near at 1230 b.p. There are also some restrictioin sites at the two sides of our target protein, provided for future team to utilize the intein tag.
  
  

Revision as of 19:46, 25 September 2018


T7 Promoter+RBS+Leucocyclicin Q+intein+CBD

NCTU_Formosa 2018 designed a composite part encoding the Leucocyclicin Q sequence (BBa_K2599006), and then combined with a T7 promoter (BBa_I712074), a lac operator (K1624002), a ribosome binding site (BBa_B0034), intein and chintin binding domain (CBD). Further information of our peptide can be found on our design page.



Figure 1. Composite part of Leucocyclicin Q


Introduction

Leucocyclicin Q, produced by Leuconostoc mesenteroides TK41401, possesses antimicrobial activity against broad range of bacteria srains, and particularly shows strong activity against Bacillus coagulans. Leucocyclicin Q was proved to be a cyclic bacteriocin in which N and C termini are bound to each other, and its structure is very similar to Lactocyclicin Q. Furthermore, it shows high stability against different pH value or heat stress.


Mechanism of Leucocyclicin Q

The bacteriocins inhibit their target organisms through pore formation. Though the mechanism of each inhibition is vary from species to species, the general process is conserved. To see more details, please search for our project page.



Features of Leucocyclicin Q

1. Species Specific

Bacteriocins are antimicrobial peptides that will kill or inhibit bcterial strains closely related or non-related to produced bacteria, but will not harm the bacteria themselves by specific immunity proteins. The organisims that Leucocyclicin Q targets including Enterococcus faecalis, Bacillus subtilis, Bacillus coagulans, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC209 bactibase].

2. Eco-friendly

Since Leucocyclicin Q is a polypeptide naturally produced by bacteria itself and can inhibit other bacteria without much environment impact. It don't pose threat to other organisms like farm animals or humans. Therefore, this toxin will not cause safety problem.

3. Biodegradable

Leucocyclicin Q is a short peptide that will degrade in a short time. After degradation, this antibacterial peptide is harmless to our environment.


Experiment Result

Cloning

We conbined our toxic gene to pSB1C3 backbone by the two restriction sites, EcoRI and SpeI, and conducted PCR to check the size of our part. The Leucocyclicin Q sequence length is around 186 b.p. For the composite part, the sequence length should be near at 1230 b.p. There are also some restrictioin sites at the two sides of our target protein, provided for future team to utilize the intein tag.


Figure 2. PCR product


Expressing

We chose E. coli 2566 strain to express our antibacterial peptides. The expression of Leucocyclicin Q fused with intein was induced by IPTG in E. coli , and intein-enterocin B specifically bound to the column through chitin binding domain would be purified.


Figure 3 SDS

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1094
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 817
    Illegal AgeI site found at 907
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 737


Reference