Difference between revisions of "Part:BBa K4323002:Design"
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(1) Péchy-Tarr, M.; Bruck, D. J.; Maurhofer, M.; Fischer, E.; Vogne, C.; Henkels, M. D.; Donahue, K. M.; Grunder, J.; Loper, J. E.; Keel, C. Molecular Analysis of a Novel Gene Cluster Encoding an Insect Toxin in Plant-Associated Strains of Pseudomonas Fluorescens. Environmental Microbiology 2008, 10 (9), 2368–2386 | (1) Péchy-Tarr, M.; Bruck, D. J.; Maurhofer, M.; Fischer, E.; Vogne, C.; Henkels, M. D.; Donahue, K. M.; Grunder, J.; Loper, J. E.; Keel, C. Molecular Analysis of a Novel Gene Cluster Encoding an Insect Toxin in Plant-Associated Strains of Pseudomonas Fluorescens. Environmental Microbiology 2008, 10 (9), 2368–2386 | ||
Latest revision as of 23:53, 6 October 2022
Truncated FitD
Figure 1. A schematic representation of Truncated FitD.
Design Notes
We use bioinformatics tools and literature to determine possible regions to truncate and to keep. Since most transmembrane domains are located within the core 1300-2200 bp, we decide to use this as our design.
Issues and goals
The problem, FitD is too big! It is 9009 bp to produce over 320,000 kDa protein. The large size makes it expensive to synthesize, difficult to clone, prone to mutation and even requires special considerations when performing SDS-PAGE analysis. From this, we set the goal of removing 2/3 of the sequence and retaining activity.
Initial investigation
From the Uniprot, a TcdA_TcdB_pore forming domain from amino acid position (1620-2278) was found in B2L231_9PSED. Further reading, we came across an article detailing a reduction in the toxicity resulting from the FitA-FitH gene cluster when the central region of FitD was deleted.(1) This let us narrow the search to the window by about half.
Homology modelling
Using SWISS-MODEL. We made a homology model of the P. protogens FitD against two other related toxins. The first is an electron microscopy structure of toxin A from Clostridioides difficle PDB:7pog as a template for the core (amino acids 1440-2187). The second was for amino acids (1301-1568) a putative RTX-toxin also called (MCF for “make caterpillar floppy”) PDB:6ii6. From the homology modelling, we found that the FitD protein could be rationalized as a large toxin with several active sub-domains. There is the MCF region, the toxin A region, and then the core tcdA_tcdB_pore. Ultimately we opted to keep the entire MCF region, as well as the toxin A region. We did end up removing 88 amino acids from the tcdA_tcdB_pore.
Figure 2. Swiss model of FitD using toxin A from Clostridioides difficle PDB:7pog as a template.
Secondary structure prediction
Last, we input our construct into [http://bioinf.cs.ucl.ac.uk/psipred/ PSIPRED] to see how many predicted transmembrane domains we had. The results from [http://bioinf.cs.ucl.ac.uk/psipred/ PSIPRED], found as part of the supplemental information, confirmed that our design had predicted transmembrane activity.
Source
Pseudomonas protegens
References
(1) Péchy-Tarr, M.; Bruck, D. J.; Maurhofer, M.; Fischer, E.; Vogne, C.; Henkels, M. D.; Donahue, K. M.; Grunder, J.; Loper, J. E.; Keel, C. Molecular Analysis of a Novel Gene Cluster Encoding an Insect Toxin in Plant-Associated Strains of Pseudomonas Fluorescens. Environmental Microbiology 2008, 10 (9), 2368–2386