Difference between revisions of "Part:BBa K4247010:Design"

Latest revision as of 14:07, 10 October 2022

Minispidroin_NT-2rep-CT_SnoopTag_N-6His

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 493
Illegal PstI site found at 499
Illegal PstI site found at 514
Illegal PstI site found at 568
Illegal PstI site found at 586
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 493
Illegal PstI site found at 499
Illegal PstI site found at 514
Illegal PstI site found at 568
Illegal PstI site found at 586
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 493
Illegal PstI site found at 499
Illegal PstI site found at 514
Illegal PstI site found at 568
Illegal PstI site found at 586
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 493
Illegal PstI site found at 499
Illegal PstI site found at 514
Illegal PstI site found at 568
Illegal PstI site found at 586
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.

Compared to the parts with only 2 central repeats (BBa_K4247004, BBa_K4247007), the process for achieving 4 central repeats was the same, but we used a cloning vector that had the full 4 repeats rather than only 2 as a fragment-giver vector in Golden Gate Cloning.

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.

In order to test if our protein would be expressed better with the His-tag in the N-terminus, we designed some primers and used PCR to switch the 6x His-tag from the C-terminus to the N-terminus. For this part, the N terminus 6His-tag was much better for production.

Further, we wanted to incorporate the SnoopTag with our protein such that it would enable the protein to bind to any other protein that has the complementary SnoopCatcher. The SnoopTag coding sequence was added to the C-terminus of the protein via PCR and User cloning.

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_K4247005 (Minispidroin_NT_N-6His), BBa_K4247001 (Minispidroin_2rep), BBa_K4247002 (Minispidroin_CT) and BBa_K4247008 (SnoopTag).

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

Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M.Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci U S A. 2016 Feb 2;113(5):1202-7.

@@ Line 7: / Line 7: @@
 ===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 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.
+Compared to the parts with only 2 central repeats (BBa_K4247004, BBa_K4247007), the process for achieving 4 central repeats was the same, but we used a cloning vector that had the full 4 repeats rather than only 2 as a fragment-giver vector in Golden Gate Cloning.
-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 optimised as per E.coli's codon bias.
+In order to test if our protein would be expressed better with the His-tag in the N-terminus, we designed some primers and used PCR to switch the 6x His-tag from the C-terminus to the N-terminus. For this part, the N terminus 6His-tag was much better for production.
-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.
+Further, we wanted to incorporate the SnoopTag with our protein such that it would enable the protein to bind to any other protein that has the complementary SnoopCatcher. The SnoopTag coding sequence was added to the C-terminus of the protein via PCR and User cloning.
-Since for some proteins, the expression is better with the His-tag on the N-terminus instead of the C-terminus we used primers to switch the 6x His-tag.
+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.
-Further, we wanted to incorporate the SnoopTag with our protein such that it would enable the protein to bind to any other protein that has the complementary SnoopCatcher. The SnoopTag coding sequence was added to the C-terminus of the protein via PCR and User cloning.
+[[File:MSp2repSnoop.png|600px|]]
 ===Source===
-The sequence of this composite part is obtained from the following basic parts: BBa_K4247005 (Minispidroin_NT_N-6His), BBa_K4247001 (Minispidroin_2rep), BBa_K4247002 (Minispidroin_CT) and BBa_K4247008 (SnoopTag).
+The sequence of this composite part is obtained from the following basic parts: <partinfo>BBa_K4247005</partinfo> (Minispidroin_NT_N-6His), <partinfo>BBa_K4247001</partinfo> (Minispidroin_2rep), <partinfo>BBa_K4247002</partinfo> (Minispidroin_CT) and <partinfo>BBa_K4247008</partinfo> (SnoopTag).
 ===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
+Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M.Programmable polyproteams built using twin peptide superglues. Proc Natl Acad Sci U S A. 2016 Feb 2;113(5):1202-7.