Difference between revisions of "Part:BBa K4323002:Design"
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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. | 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. | ||
| + | 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. | ||
| + | |||
| + | From the Uniprot, a TcdA_TcdB_pore forming domain from amino acid position (1620-2278) was found in B2L231_9PSED https://www.uniprot.org/uniprotkb/B2L231/entry. 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. This let us narrow the search to the window by about half. | ||
| + | |||
| + | Using SWISS-MODEL https://swissmodel.expasy.org/interactive. 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. | ||
Revision as of 00:02, 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.
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.
From the Uniprot, a TcdA_TcdB_pore forming domain from amino acid position (1620-2278) was found in B2L231_9PSED https://www.uniprot.org/uniprotkb/B2L231/entry. 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. This let us narrow the search to the window by about half.
Using SWISS-MODEL https://swissmodel.expasy.org/interactive. 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.
Source
Pseudomonas protegens