Difference between revisions of "Part:BBa K4247011:Design"

(→‎Design Notes)
(→‎Design Notes)
Line 7: Line 7:
  
 
===Design Notes===
 
===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 two repetitive parts would be inserted in between the N- and C-terminus to give the whole minispidroin protein. We decided to produce a protein similar to Minispidroin_NT-2rep-CT (composite part BBa_K247007) because higher repeats in spider silks were shown to give better fibre performance. The DNA sequence coding for the minispidroin protein was then placed 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 one and the repetitive part in another smaller 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 chosen to split the N- and C-terminus into 2 basic parts.
+
We decided to produce a protein similar to Minispidroin_NT-2rep-CT (composite part BBa_K247007) because higher repeats in spider silks were shown to give better fibre performance. 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 donating vector in Golden Gate Cloning.
  
  
−
Further, this sequence has been codon optimised as per E.coli's codon bias. 
+
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.
−
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.
+
 
 +
 
 +
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===
 
===Source===

Revision as of 15:27, 26 September 2022


Minispidroin_NT-4rep-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
    Illegal PstI site found at 721
    Illegal PstI site found at 727
  • 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
    Illegal PstI site found at 721
    Illegal PstI site found at 727
  • 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
    Illegal PstI site found at 721
    Illegal PstI site found at 727
  • 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
    Illegal PstI site found at 721
    Illegal PstI site found at 727
  • 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 one and the repetitive part in another smaller 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.


We decided to produce a protein similar to Minispidroin_NT-2rep-CT (composite part BBa_K247007) because higher repeats in spider silks were shown to give better fibre performance. 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 donating 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.


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), 2x 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