Difference between revisions of "Part:BBa K4247004:Design"

Latest revision as of 14:05, 10 October 2022

Minispidroin_NT-2rep-CT

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 469
Illegal PstI site found at 475
Illegal PstI site found at 490
Illegal PstI site found at 544
Illegal PstI site found at 562
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 469
Illegal PstI site found at 475
Illegal PstI site found at 490
Illegal PstI site found at 544
Illegal PstI site found at 562
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 469
Illegal PstI site found at 475
Illegal PstI site found at 490
Illegal PstI site found at 544
Illegal PstI site found at 562
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 469
Illegal PstI site found at 475
Illegal PstI site found at 490
Illegal PstI site found at 544
Illegal PstI site found at 562
1000
COMPATIBLE WITH RFC[1000]

Design Notes

It is difficult to synthesise the entire DNA sequence of minispidroins due to the repetitiveness of the central motifs. So, at the UCopenhagen team, we have decided to split the protein into the N-terminus and C-terminus in an expression plasmid and the repetitive part in another cloning plasmid which is easier to produce. The DNA sequence coding for the N- and C-terminus was designed to be separated by a spacer containing two BsaI sites while the repetitive (central) part of the final protein would have 2 BsaI sites on each end. In this way, the repetitive sequence was added in between the N and C terminus to get a whole protein.

The DNA sequence coding for the minispidroin protein was also contained in a pET24 expression vector having a T7 promoter,terminator and KAN resistance gene. Some proteins are expressed better if they have the His-tag in the N-terminus or vice versa. Our expression vector - pET24 (+) - has a 6x His-tag in the C-terminus.

Since the type IIS assembly compatibility system forbids the presence of a BsaI recognition site within the sequence of a part, we have chosen to split the N- and C-terminus into 2 basic parts here in the Registry. Further, this sequence was codon optimised as per E.coli's codon bias.

Source

The sequence of this composite part is obtained from the following basic parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247001 (Minispidroin_2rep) and BBa_K4247002 (Minispidroin_CT).

References

Andersson, M., Jia, Q., Abella, A. et al. Biomimetic spinning of artificial spider silk from a chimeric minispidroin. Nat Chem Biol 13, 262–264 (2017). https://doi.org/10.1038/nchembio.2269

Strickland, M., Tudorica, V., Řezáč, M. et al. Conservation of a pH-sensitive structure in the C-terminal region of spider silk extends across the entire silk gene family. Heredity 120, 574–580 (2018). https://doi.org/10.1038/s41437-018-0050-9

@@ Line 7: / Line 7: @@
 ===Design Notes===
-The DNA sequence coding for the N- and C-terminus would be separated by a spacer containing a BsaI site and the repetitive part of the protein would have 2 BsaI sites on each end such that when Golden Gate Cloning was performed, the repetitive part would be inserted in between the N- and C-terminus to give the whole minispidroin protein. The DNA sequence coding for the minispidroin protein will be contained in a pET24 expression vector containing a T7 promoter, terminator and a 6x His-tag following the C-terminus of the protein to facilitate protein purification.
+It is difficult to synthesise the entire DNA sequence of minispidroins due to the repetitiveness of the central motifs. So, at the UCopenhagen team, we have decided to split the protein into the N-terminus and C-terminus in an expression plasmid and the repetitive part in another cloning plasmid which is easier to produce. The DNA sequence coding for the N- and C-terminus was designed to be separated by a spacer containing two BsaI sites while the repetitive (central) part of the final protein would have 2 BsaI sites on each end. In this way, the repetitive sequence was added in between the N and C terminus to get a whole protein.
-However, since the type IIS assembly compatibility system forbids the presence of a BsaI recognition site within the sequence of a part, we have chose to split the N- and C-terminus into 2 basic parts.
+The DNA sequence coding for the minispidroin protein was also contained in a pET24 expression vector having a T7 promoter,terminator and KAN resistance gene. Some proteins are expressed better if they have the His-tag in the N-terminus or vice versa. Our expression vector - pET24 (+) - has a 6x His-tag in the C-terminus.
-Further, this sequence has been codon optimized as per E.coli's codon bias.
+Since the type IIS assembly compatibility system forbids the presence of a BsaI recognition site within the sequence of a part, we have chosen to split the N- and C-terminus into 2 basic parts here in the Registry. Further, this sequence was codon optimised as per E.coli's codon bias.
-It is difficult to synthesise the DNA sequence coding for the central part of the minispidroin due to its repetitiveness. So, at the UCopenhagen team, we have decided to split the protein into the N-terminus and C-terminus in one and the repetitive part in another plasmid. Using Golden Gate Assembly, the repetitive part can be inserted in between the N and C terminus to get the coding sequence of the entire minispidroin protein. In this way, any sequence can be added in between the N and C terminus to get a whole protein.
+[[File:2rep.png|600px]]
 ===Source===
-The sequence of this composite part is obtained from the following basic parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247001 (Minispidroin_2rep) and BBa_K4247002 (Minispidroin_CT).
+The sequence of this composite part is obtained from the following basic parts: <partinfo>BBa_K4247000</partinfo> (Minispidroin_NT), <partinfo>BBa_K4247001</partinfo> (Minispidroin_2rep) and <partinfo>BBa_K4247002</partinfo> (Minispidroin_CT).
 ===References===