Difference between revisions of "Part:BBa K4229071"

Latest revision as of 17:34, 11 October 2022

BMC-T1_F8TAG

BMC-T1-F8TAG Short description: The F8TAG variant of BMC-T1(BBa_K4229037) was created using site-directed mutagenesis, aiming to insert a non-canonical amino acid at position 8 of the peptide sequence by amber stop codon suppression. The BMC-T1_F8TAG can only be completely synthesized upon successful amber stop codon suppression. Hence, BMC-T1_F8TAG can be used to create a ncAA modified shell protein for the assembly of a synthetic carboxysome.

Usage: Besides the well-known 20 canonical amino acids, a variety of non-canonical amino acids can be used to modify proteins. One way of incorporating non-canonical amino acids is via the amber stop codon suppression technology. BMC-T1_F8TAG is a mutated version of the BMC-T1 protein, necessary to form the synthetic BMC of the myxobacterium Haliangium ochraceum. Without successful incorporation, a non-functional version of BMC-T1 will be translated, and the synthetic carboxysome cannot form. However, after successful incorporation of the non-canonical amino acid, translation will go on as normal and the synthetic carboxysome can form.

Characterization: The incorporation of non-canonical amino acids into BMC-T1_ F8TAG was characterized using SDS-PAGE, Western blot, and antibody decoration. As an orthogonal translation system, an additional aminoacyl-tRNA synthetase and its corresponding tRNA are required, which in this case correspond to the pAzF tRNA synthetase (pAzFRS), which incorporates 4-azido-l-phenylalanine.

Figure text: SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 8, 35, 78 and 96 of the BMC-T1 protein. The cells grew until OD 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used for orientation. To purify the protein, the His-Spin Protein Miniprep from Zymo Research was used.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 706
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 33
Illegal AgeI site found at 510
Illegal AgeI site found at 618
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
-Short Description:
+BMC-T1-F8TAG
-The F8X variant of BMC-T1(BBa_K4229037) was created using site-directed mutagenesis, aiming to insert a non-canonical amino acid at position 8 of the peptide sequence by amber stop codon. The BMC-T1_F8X can only be completely synthesized upon successful amber stop codon suppression. Hence, BMC-T1_F8X can be used to create a ncAA modified shell protein for the assembly of a synthetic carboxysome.
+Short description:
+The F8TAG variant of BMC-T1(BBa_K4229037) was created using site-directed mutagenesis, aiming to insert a non-canonical amino acid at position 8 of the peptide sequence by amber stop codon suppression. The BMC-T1_F8TAG can only be completely synthesized upon successful amber stop codon suppression. Hence, BMC-T1_F8TAG can be used to create a ncAA modified shell protein for the assembly of a synthetic carboxysome.
 Usage:
-Besides the well-known 20 canonical amino acids, there is a variety of non-canonical amino acids which can be used to further modify proteins. One way of incorporating non-canonical amino acids is via the amber stop codon suppression technology. This part BMC-T1_F8X, is a mutated version of the BMC-T1 protein, necessary to form the synthetic BMC of the myxobacterium Haliangium ochraceum. Without successful incorporation, a non-functional version of BMC-T1 will be translated, and the synthetic carboxysome cannot form. However, after successful incorporation of the non-canonical amino acid, translation will go on as normal and the synthetic carboxysome can form.
+Besides the well-known 20 canonical amino acids, a variety of non-canonical amino acids can be used to modify proteins. One way of incorporating non-canonical amino acids is via the amber stop codon suppression technology. BMC-T1_F8TAG is a mutated version of the BMC-T1 protein, necessary to form the synthetic BMC of the myxobacterium Haliangium ochraceum. Without successful incorporation, a non-functional version of BMC-T1 will be translated, and the synthetic carboxysome cannot form. However, after successful incorporation of the non-canonical amino acid, translation will go on as normal and the synthetic carboxysome can form.
 Characterization:
-The incorporation of non-canonical amino acids into BMC-T1_F8X was characterized using SDS-PAGE, Western blot, and antibody decoration.  As an orthogonal translation system, an additional aminoacyl-tRNA synthetase and its corresponding tRNA are required, which in this case correspond to the pAzf synthetase, which incorporates 4-azido-l-phenylalanine.
+The incorporation of non-canonical amino acids into BMC-T1_ F8TAG was characterized using SDS-PAGE, Western blot, and antibody decoration.  As an orthogonal translation system, an additional aminoacyl-tRNA synthetase and its corresponding tRNA are required, which in this case correspond to the pAzF tRNA synthetase (pAzFRS), which incorporates 4-azido-l-phenylalanine.
+[[File:T1MutWestern.jpg|800px|thumb|left|Figure text: SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 8, 35, 78 and 96 of the BMC-T1 protein. The cells grew until OD 0.4, following the addition of arabinose and a growth period of 2 hours. After 2 hours, the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used for orientation. To purify the protein, the His-Spin Protein Miniprep from Zymo Research was used. ]]
-[[File:T1MutWestern.jpg|800px|thumb|left|Figure text: SDS-PAGE with whole cell lysis confirming the incorporation of pAzF at position 8, 35, 78 and 96 of the BMC-T1 protein. The cells grew until OD 0.4, following the addition of arabinose and a growth period of 2 h. After 2 hours the cells were treated with IPTG and grew 24 hours before harvesting by centrifugation. The BlueClassic Prestained Protein Marker, 10-180 kDa from Jena Bioscience was used for orientation. To purify the protein, the His-Spin Protein Miniprep from Zymo Research was used. ]]