Difference between revisions of "Part:BBa K4813000"

Revision as of 09:59, 7 October 2023

dTomato fluoresence protein Codon Optimized for E coli

This part encodes a protein called dTomato, which is a red fluorescence protein. It forms a dimer and has a molecular weight of around 27.0 kDa. This protein originates from a species called Discosoma [1].

To enhance its performance for our project, we used IDT codon optimization tools to optimize the sequence. We incorporated this optimized sequence into two composite parts. One part served as a reporter gene for detecting formaldehyde with pFrmR formaldehyde sensing promoter (BBa_K4813003), while the other part (BBa_K4813005) acted as a positive control, where the expression of dTomato was driven by a strong constitutive promoter (BBa_J23100).

Reference

[1] dTomato at fpbase. FPbase. Accessed 20 June 2023 https://www.fpbase.org/protein/dtomato/

[2] tdTomato at fpbase. FPbase. Accessed 20 June 2023 https://www.fpbase.org/protein/tdtomato/

Usage and Biology

Comparing the colouration of optimized dTomato BBa_K4813000 and tdTomato BBa_K4813001</b>

While the fluorescence protein database indicates that tdTomato exhibits superior fluorescence emission compared to dTomato [2], we don't know if it also applies to their chromoprotein properties. Given that our project focuses on developing a device to monitor formaldehyde levels that can be easily used by the general population without specialized equipment, we are interested in finding a chromoprotein that produces a more obvious color visible to the naked eye.

To address this, we have generated two composite parts, one expressing the dTomato part (BBa_K4813002) and the other expressing tdTomato (BBa_K4813004), and subsequently compared their colors as observed by the naked eye.

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The bacterial colonies expressing dTomato on the LB/Amp agar plates (Fig. 1) exhibited a deeper red color compared to those expressing tdTomato. We hypothesized that it is due to the larger size of tdTomato (which is composed of two dTomato proteins) may require more energy for expression by the E. coli cells. As a result, this increased energy demand potentially leads to a slower growth rate of the cells expressing tdTomato and a lower overall protein expression level within the colonies.

Consequently, we chose to use this part for the final assay, as it proved to be more obvious in detecting the change of colour in E. coli with the presence of formaldehyde.

Sequence and Features