Difference between revisions of "Part:BBa K2599013"

Revision as of 18:04, 20 September 2018

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). Further information of our peptide can be found on our design page.

Figure 1 biobrick picture

Introduction

Mechanism of Enterocin 96

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 Enterocin 96 targets including Enterococcus faecalis, Bacillus subtilis, Listeria monocytogenes, etc. More target organisms can be found on [http://bactibase.hammamilab.org/BAC149 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 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.

Figure 2 PCR

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1064
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 787
Illegal AgeI site found at 877
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 707

Reference

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2599013 short</partinfo>
-NCTU_Formosa 2018 designed a composite part encoding the Enterocin 96 sequence [https://parts.igem.org/Part:BBa_K2599004 (BBa_K2599004)], and then combined with a T7 promoter [https://parts.igem.org/Part:BBa_I712074 (BBa_I712074)], a lac operator [https://parts.igem.org/Part:BBa_K1624002 (K1624002)], a ribosome binding site [https://parts.igem.org/Part:BBa_B0034 (BBa_B0034)], intein and chintin binding domain (CBD). Further information of our peptide can be found on our design page.
+NCTU_Formosa 2018 designed a composite part encoding the Lacticin Z sequence [https://parts.igem.org/Part:BBa_K2599005 (BBa_K2599005)], and then combined with a T7 promoter [https://parts.igem.org/Part:BBa_I712074 (BBa_I712074)], a lac operator [https://parts.igem.org/Part:BBa_K1624002 (K1624002)], a ribosome binding site [https://parts.igem.org/Part:BBa_B0034 (BBa_B0034)], intein and chintin binding domain (CBD). Further information of our peptide can be found on our design page.
@@ Line 11: / Line 11: @@
 <p style="padding-top:20px;font-size:20px"><b>Introduction</b></p>
-Enterocin 96, is a classII bacteriocin produced by <i>Enterococcus faecalis</i>. It has strong activity against most gram-positive strains but almost no activity against gram-negative strains, such as <i>Escherichia coli</i>. The antimicrobial spectrum is relatively wide compared with other bacteriocins from lactic acid bacteria.
@@ Line 18: / Line 17: @@
 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.
-Enterocin 96, like most of the class IIa bacteriocins, acts on the cytoplasmic membrane of gram-positive cells. It will bind to the receptor leading to an irreversible opening of an interinsic channel, allowing to form a pore, and therefore dissipate the transmembrane elctrical potential.
-<p style="padding-top:10px;font-size:20px;"><b>Features of Enterocin 96</b></p>
+<p style="padding-top:10px;font-size:20px;"><b>Features of Lacticin Z</b></p>
 <p style="padding-top:16px;font-size:16px"><b>1. Species Specific</b></p>
@@ Line 28: / Line 27: @@
 <p style="padding-top:16px;font-size:16px"><b>2. Eco-friendly</b></p>
-Since enterocin 96 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.
+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.
 <p style="padding-top:16px;font-size:16px"><b>3. Biodegradable</b></p>
-Enterocin 96 is a short peptide that will degrade in a short time. After degradation, this antibacterial peptide is harmless to our environment.
+Lacticin Z is a short peptide that will degrade in a short time. After degradation, this antibacterial peptide is harmless to our environment.
@@ Line 39: / Line 38: @@
 ===Cloning===
-We conbined our toxic gene to pSB1C3 backbone and conducted PCR to check the size of our part. The enterocin 96 sequence length is around 219 b.p. For the composite part, the sequence length should be near at 1263 b.p.
+We conbined our toxic gene to pSB1C3 backbone 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.
@@ Line 48: / Line 47: @@
 ===Expressing===
-We chose <i> E. coli </i> 2566 strain to express our antibacterial peptides. The expression of enterocin 96 fused with intein was induced by IPTG in <i> E. coli </i>, and intein-enterocin B specifically bound to the column through chitin binding domain would be purified.
+We chose <i> E. coli </i> 2566 strain to express our antibacterial peptides. The expression of Lacticin Z fused with intein was induced by IPTG in <i> E. coli </i>, and intein-enterocin B specifically bound to the column through chitin binding domain would be purified.