Difference between revisions of "Part:BBa K3114014:Design"
 
(One intermediate revision by the same user not shown)
Line 2: Line 2:
 
<partinfo>BBa_K3114014 short</partinfo>
 
<partinfo>BBa_K3114014 short</partinfo>
 
<partinfo>BBa_K3114014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3114014 SequenceAndFeatures</partinfo>
 +
 
===Design Notes===
 
===Design Notes===
−
ICARUS was designed to enable purification of large proteins with strong electrostatic potential in their binding pockets using a 6xHis-tag. The part is designed to be attached in-frame with a protein coding region at the C-terminal, which can be accomplished via Golden Gate reaction using MoClo assembly standard overhangs. The predicted structure of the universal spacer, modelled using <i>ab initio</i> and Rosetta comparative modelling creates a "helix-turn-helix-turn-helix" motif. SacII restriction sites are positioned at the start of the spacer and after the 6xHis-tag for removal of the 6x His-tag at the DNA level. In addition, a thrombin proteolytic site exists in the first turn of the predicted motif for 6xHis-tag removal at the protein level. The second turn in the predicted motif is filled with aspartic acid residues to repel electronegative forces, if used with a protein that has a binding pocket that is highly electronegative. This part also includes a double stop codon and a double terminator <a href="https://parts.igem.org/Part:BBa_B0014">(BBa_B0014).</a> Codons were alternated to avoid tRNA depletion and were optimized based on E. coli molecular machinery.
+
[[Image:T--Calgary--ICARUS-Predicted-Registry.jpeg|200px|thumb|right|Figure 2A. Hypothesized structure of ICARUS containing a "helix-turn-helix-turn-helix" structure.]][[Image:T--Calgary--ICARUS-ab-initio-Registry.gif|200px|thumb|right|Figure 2B. Structure of ICARUS determined by <i>ab initio</i> structural prediction modelling.]]
 +
 
 +
When designing circuits for the inducible expression and purification of our chlorophyll degradation pathway enzymes ([https://parts.igem.org/Part:BBa_K3114024 (CBR: BBa_K3114024)], [https://parts.igem.org/Part:BBa_K3114025 (7-HCAR: BBa_K3114025)], [https://parts.igem.org/Part:BBa_K3114026 (SGR: BBa_K3114026)], [https://parts.igem.org/Part:BBa_K3114027 (PPH: BBa_K3114027)]) we found that 7-HCAR was previously recombinantly expressed in <i>E. coli</i>, but catalytically inactive (Meguro et al., 2011). Through electrostatic interaction modeling of 7-HCAR, which can be read about [https://2019.igem.org/Team:Calgary/Model/ICARUS (here)], it was hypothesized that the strong electronegative binding pocket prevented 6xHis tag purification. Additionally, previous attempts to recombinantly express and purify PPH via 6xHis tag and a maltose binding protein fusion (MBP) were unsuccessful (Guyer, Salinger, Krügel, & Hörtensteiner, 2017).
 +
 
 +
To overcome these problems we sought to create a sequence that could fold between the core protein and the 6xHis tag to provide enough space that would allow for the functionality of both the protein and the purification tag.
 +
 
 +
ICARUS was designed to enable purification of large proteins with strong electrostatic potential in their binding pockets using a 6xHis-tag. The part is designed to be attached in-frame with a protein coding region at the C-terminal, which can be accomplished via Golden Gate reaction using MoClo assembly standard overhangs. The predicted structure of the universal spacer, modelled using <i>ab initio</i> and Rosetta comparative modelling creates a "helix-turn-helix-turn-helix" motif. SacII restriction sites are positioned at the start of the spacer and after the 6xHis-tag for removal of the 6x His-tag at the DNA level. In addition, a thrombin proteolytic site exists in the first turn of the predicted motif for 6xHis-tag removal at the protein level. The second turn in the predicted motif is filled with aspartic acid residues to repel electronegative forces, if used with a protein that has a binding pocket that is highly electronegative. However, PPH has electropositive forces within its native structure and was still shown to be purified using ICARUS fusion and was catalytically active. This part also includes a double stop codon and a double terminator [https://parts.igem.org/Part:BBa_B0014 (BBa_B0014).] Codons were alternated to avoid tRNA depletion and were optimized based on <i>E. coli</i> molecular machinery.
 +
 
 
===Source===
 
===Source===
−
wip
+
This part was synthesized.
 +
 
 
===References===
 
===References===
 +
Guyer, L., Salinger, K., Krügel, U., & Hörtensteiner, S. (2017). Catalytic and structural properties of pheophytinase, the phytol esterase involved in chlorophyll breakdown. Journal of Experimental Botany, 69(4), 879–889. doi: 10.1093/jxb/erx326
 +
 +
Meguro, M., Ito, H., Takabayashi, A., Tanaka, R., & Tanaka, A. (2011). Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis. The Plant Cell, 23(9), 3442–3453. doi: 10.1105/tpc.111.089714
 +
 +
Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765

Latest revision as of 22:11, 12 December 2019

ICARUS spacer + 6XHis tag + double terminator for purification of highly electronegative proteins


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 215
    Illegal XhoI site found at 318
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 321

Design Notes

Figure 2A. Hypothesized structure of ICARUS containing a "helix-turn-helix-turn-helix" structure.
Figure 2B. Structure of ICARUS determined by ab initio structural prediction modelling.

When designing circuits for the inducible expression and purification of our chlorophyll degradation pathway enzymes ((CBR: BBa_K3114024), (7-HCAR: BBa_K3114025), (SGR: BBa_K3114026), (PPH: BBa_K3114027)) we found that 7-HCAR was previously recombinantly expressed in E. coli, but catalytically inactive (Meguro et al., 2011). Through electrostatic interaction modeling of 7-HCAR, which can be read about (here), it was hypothesized that the strong electronegative binding pocket prevented 6xHis tag purification. Additionally, previous attempts to recombinantly express and purify PPH via 6xHis tag and a maltose binding protein fusion (MBP) were unsuccessful (Guyer, Salinger, Krügel, & Hörtensteiner, 2017).

To overcome these problems we sought to create a sequence that could fold between the core protein and the 6xHis tag to provide enough space that would allow for the functionality of both the protein and the purification tag.

ICARUS was designed to enable purification of large proteins with strong electrostatic potential in their binding pockets using a 6xHis-tag. The part is designed to be attached in-frame with a protein coding region at the C-terminal, which can be accomplished via Golden Gate reaction using MoClo assembly standard overhangs. The predicted structure of the universal spacer, modelled using ab initio and Rosetta comparative modelling creates a "helix-turn-helix-turn-helix" motif. SacII restriction sites are positioned at the start of the spacer and after the 6xHis-tag for removal of the 6x His-tag at the DNA level. In addition, a thrombin proteolytic site exists in the first turn of the predicted motif for 6xHis-tag removal at the protein level. The second turn in the predicted motif is filled with aspartic acid residues to repel electronegative forces, if used with a protein that has a binding pocket that is highly electronegative. However, PPH has electropositive forces within its native structure and was still shown to be purified using ICARUS fusion and was catalytically active. This part also includes a double stop codon and a double terminator (BBa_B0014). Codons were alternated to avoid tRNA depletion and were optimized based on E. coli molecular machinery.

Source

This part was synthesized.

References

Guyer, L., Salinger, K., Krügel, U., & Hörtensteiner, S. (2017). Catalytic and structural properties of pheophytinase, the phytol esterase involved in chlorophyll breakdown. Journal of Experimental Botany, 69(4), 879–889. doi: 10.1093/jxb/erx326

Meguro, M., Ito, H., Takabayashi, A., Tanaka, R., & Tanaka, A. (2011). Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis. The Plant Cell, 23(9), 3442–3453. doi: 10.1105/tpc.111.089714

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765