Difference between revisions of "Part:BBa K4137003"

Latest revision as of 03:40, 10 October 2022

Chi18h8-YebF Fusion Protein

This part consists of the secretion tag YebF (BBa_K2922002), a GS Linker (BBa_J18921), a Chi18H8 coding sequence, and a 6xHis tag. The sequence is reversed in our experimentation, but for documentation purposes, it is entered in the forward directionality.

Fig.1 Chi18H8 fused with yebF by a GS linker.

Construct Design

During the gBlocks fragment synthesis stage of our construct design, we were forced to split Chi18H8 chitinase along the restriction sites ClaI due to errors in IDT gblock fragment synthesis, in which the first half would have cut sites at EcoRI and ClaI and the second half would have cut sites at ClaI and SpeI. We first ligated the first half of chitinase onto our pSB1C3 backbone before attaching the second half. The full Chi18H8 construct would have EcoRI and SpeI cut sites.

Fig.2 Chi18h8 first half with E-C cut sites and second half with C-S cut sites.

Our goal is to secrete Chi18H8 extracellularly via the SEC secretion system, so we fused Chi18H8 with yebF, a N-terminal peptide secretion tag that facilitates the movement of chitinase through the periplasm and into the extracellular medium. Upon secreting our protein of interest into the extracellular medium, yebF would be cleaved off. A flexible glycine-serine linker (GS) is inserted between the two genes to stabilize our fusion protein.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1492
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1005
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 552
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4137003 short</partinfo>
-This part consists of the secretion tag YebF (BBa_K2922002), a GS Linker (BBa_J18921), a chi18h8 coding sequence, and a 6xHis tag. The sequence is reversed in our experimentation, but for documentation purposes, it is entered in the forward directionality.
+This part consists of the secretion tag YebF (BBa_K2922002), a GS Linker (BBa_J18921), a Chi18H8 coding sequence, and a 6xHis tag. The sequence is reversed in our experimentation, but for documentation purposes, it is entered in the forward directionality.
+[[File:his-chitinase-gs-yebf-design.png|800px|thumb|center|Fig.1 Chi18H8 fused with yebF by a GS linker.]]
 ===Construct Design===
-During the gBlocks fragment synthesis stage of our construct design, we were forced to split Chi18h8 chitinase along the restriction sites ClaI due to errors in IDT gblock fragment synthesis, in which the first half would have cut sites at EcoRI and ClaI and the second half would have cut sites at ClaI and SpeI. We first ligated the first half of chitinase onto our pSB1C3 backbone before attaching the second half. The full Chi18h8 construct would have EcoRI and SpeI cut sites.
+During the gBlocks fragment synthesis stage of our construct design, we were forced to split Chi18H8 chitinase along the restriction sites ClaI due to errors in IDT gblock fragment synthesis, in which the first half would have cut sites at EcoRI and ClaI and the second half would have cut sites at ClaI and SpeI. We first ligated the first half of chitinase onto our pSB1C3 backbone before attaching the second half. The full Chi18H8 construct would have EcoRI and SpeI cut sites.
+[[File:his-chitinase-gs-yebf-half-cut.png|800px|thumb|center|Fig.2 Chi18h8 first half with E-C cut sites and second half with C-S cut sites.]]
+Our goal is to secrete Chi18H8 extracellularly via the SEC secretion system, so we fused Chi18H8 with yebF, a N-terminal peptide secretion tag that facilitates the movement of chitinase through the periplasm and into the extracellular medium. Upon secreting our protein of interest into the extracellular medium, yebF would be cleaved off. A flexible glycine-serine linker (GS) is inserted between the two genes to stabilize our fusion protein.
 ===Sequence and Features===