Part:BBa_K2599013
T7 Promoter+RBS+Lacticin Z+intein+CBD
NCTU_Formosa 2018 designed a composite part encoding the Lacticin Z sequence (BBa_K2599005), 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) (BBa_K1465230). Further information of our peptide can be found on our design page.
Figure 1. Composite part of Lacticin Z
Introduction
Lacticin Z, produced by Lactoccus lactis QU 14, has no leader sequence or signal peptide. Also, it exhibits a nanomolar range of MICs against various gram-positve bacteria. Consequently, Lacticin Z indeed shows the best antibacterial activity in our experiment.
Mechanism of Lacticin Z
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 Lacticin Z
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 Lacticin Z targets including Enterococcus faecium, Bacillus subtilis, Bacillus coagulans, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC170 bactibase].
2. Eco-friendly
Since Lacticin Z 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
Lacticin Z 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 Lacticin Z sequence length is around 153 b.p. For the composite part, the sequence length should be near at 1197 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 Lacticin Z 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1064
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 787
Illegal AgeI site found at 877 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 707
Reference
| None |