Difference between revisions of "Part:BBa K4813000"

Revision as of 08:50, 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).

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 pronounced color visible to the naked eye.

===Comparing the colouration of [i]E. coli[/i] colonies expressing optimized dTomato BBa_K4813000 and tdTomato BBa_K4813001 RFP

File:Https://static.igem.wiki/teams/4813/wiki/experiments/plate-expressing-j23100-dtom.jpeg

Figure 1: The [i]E. coli[/i] colonies expressing dTomato BBa_K4813000 and tdTomato BBa_K4813001 RFP. Cultured on LB/Amp agar plates. White colonies were formed possibly due to incomplete digestion of pUC19 plasmids before the assembly process.

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.

Consequently, we opted to use dTomato for the final assay, as it proved to be more effective in detecting the presence of formaldehyde.

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/

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4813000 short</partinfo>
-The coding sequence mentioned encodes a protein called dTomato, which produces orange fluorescence. It forms a dimer and has a molecular weight of around 27.0 kDa. This protein originates from a species called Discosoma.
+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 (pFrmR formaldehyde sensing), while the other part acted as a positive control, where the expression of dTomato was driven by a strong and consistent promoter J23100.
+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 (<partinfo>BBa_K4813003</partinfo>), while the other part (<partinfo>BBa_K4813005</partinfo>) acted as a positive control, where the expression of ''dTomato'' was driven by a strong constitutive promoter (<partinfo>BBa_J23100</partinfo>).
-Despite previous literature suggesting that tdTomato exhibits better expression and brighter light emission compared to dTomato, our experimental results demonstrated that dTomato displayed a significantly brighter red color than tdTomato in our positive controls. As a result, we selected dTomato for the final assay to effectively detect the presence of formaldehyde.
+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 pronounced color visible to the naked eye.
+===Comparing the colouration of [i]E. coli[/i] colonies expressing optimized dTomato <partinfo>BBa_K4813000</partinfo> and tdTomato <partinfo>BBa_K4813001</partinfo> RFP
+[[Image:https://static.igem.wiki/teams/4813/wiki/experiments/plate-expressing-j23100-dtom.jpeg|450px|thumb||'''Figure 1:''' The [i]E. coli[/i] colonies expressing dTomato <partinfo>BBa_K4813000</partinfo> and tdTomato <partinfo>BBa_K4813001</partinfo> RFP. Cultured on LB/Amp agar plates. White colonies were formed possibly due to incomplete digestion of pUC19 plasmids before the assembly process.]]
+To address this, we have generated two composite parts, one expressing the dTomato part (<partinfo>BBa_K4813002</partinfo>) and the other expressing tdTomato (<partinfo>BBa_K4813004</partinfo>), and subsequently compared their colors as observed by the naked eye.
+Consequently, we opted to use dTomato for the final assay, as it proved to be more effective in detecting the presence of formaldehyde.
+===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/
 <!-- Add more about the biology of this part here