Difference between revisions of "Part:BBa K4813005"

Revision as of 11:09, 7 October 2023

J23100 - dTomato red chromoprotein strong expression construct

This composite component comprises several elements, including a constitutive strong promoter (BBa_J23100), a strong ribosome binding site (BBa_B0034), an E. coli codon-optimized chromoprotein and red fluorescent protein dTomato coding sequence (BBa_K4813000), and a strong double terminator (BBa_B0015). Additionally, the 5' and 3' ends of the composite part feature a 20 base pair overlap sequence designed for the pUC19 EcoRI restriction site for NEBuilder HiFi assembly.

Our project aims to create a formaldehyde-sensing reporter. This composite part served three purposes:

1. Comparing expression levels of dTomato and tdTomato chromoproteins. 2. Providing a reference for expression levels in our formaldehyde-sensing reporter (BBa_K4813002). 3. Acting as a positive control for the cloning process.

Usage and Biology

Comparing the colouration of optimized dTomato BBa_K4813000 and tdTomato BBa_K4813001

Although the fluorescence protein database suggests that tdTomato has better fluorescence emission compared to dTomato [2], it does not provide information about their chromoprotein properties. Since our project aims to create a user-friendly device for monitoring formaldehyde levels without requiring specialized equipment, we are looking for a chromoprotein that produces a more visible color to the naked eye.

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

File:Logo-png.jpg

The bacterial colonies expressing dTomato on the LB/Amp agar plates (Fig. 1) exhibited a deeper red color compared to those expressing tdTomato (Fig. 2) . 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. To confirm our hypothesis, further investigation will be necessary.

Consequently, we chose this part for the functional 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 27
Illegal NheI site found at 50
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_K4813005 short</partinfo>
-This composite component comprises several elements, including a constitutive strong promoter (J23100), a strong ribosome binding site (B0034), an E. coli codon-optimized chromoprotein and red fluorescent protein dTomato coding sequence (K4813000), and a strong double terminator (B0015). Additionally, the 5' and 3' ends of the composite part feature a 20 base pair overlap sequence designed for the pUC19 EcoRI restriction site for NEBuilder HiFi assembly.
+This composite component comprises several elements, including a constitutive strong promoter (<partinfo>BBa_J23100</partinfo>), a strong ribosome binding site (<partinfo>BBa_B0034</partinfo>), an ''E. coli'' codon-optimized chromoprotein and red fluorescent protein dTomato coding sequence (<partinfo>BBa_K4813000</partinfo>), and a strong double terminator (<partinfo>BBa_B0015</partinfo>). Additionally, the 5' and 3' ends of the composite part feature a 20 base pair overlap sequence designed for the pUC19 EcoRI restriction site for NEBuilder HiFi assembly.
-Our project aims to create a formaldehyde sensing reporter. This composite part served three purposes:
+Our project aims to create a formaldehyde-sensing reporter. This composite part served three purposes:
-Comparing expression levels of dTomato and tdTomato chromoproteins.
+. Comparing expression levels of dTomato and tdTomato chromoproteins.
-Providing a reference for expression levels in our formaldehyde sensing reporter.
+. Providing a reference for expression levels in our formaldehyde-sensing reporter (<partinfo>BBa_K4813002</partinfo>).
-Acting as a positive control to ensure successful cloning.
+. Acting as a positive control for the cloning process.
-Literature suggests that tdTomato should have higher expression levels when expressed in E. coli colonies compared to dTomato. However, our results showed that E. coli colonies expressing dTomato had a brighter red color even under naked eyes. Therefore, our project proceeded with using dTomato constructs to increase the sensitivity of our formaldehyde reporter.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+<b>Comparing the colouration of optimized dTomato <partinfo>BBa_K4813000</partinfo> and tdTomato <partinfo>BBa_K4813001</partinfo></b>
+Although the fluorescence protein database suggests that tdTomato has better fluorescence emission compared to dTomato [2], it does not provide information about their chromoprotein properties. Since our project aims to create a user-friendly device for monitoring formaldehyde levels without requiring specialized equipment, we are looking for a chromoprotein that produces a more visible color to the naked eye.
+To address this, we have generated two composite parts, one expressing the ''dTomato'' coding sequence(<partinfo>BBa_K4813002</partinfo>) and the other expressing ''tdTomato'' coding sequence (<partinfo>BBa_K4813004</partinfo>), and subsequently compared their colors as observed by the naked eye.
+[[Image:logo-png.jpg]]
+The bacterial colonies expressing dTomato on the LB/Amp agar plates (Fig. 1) exhibited a deeper red color compared to those expressing tdTomato (Fig. 2) . 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. To confirm our hypothesis, further investigation will be necessary.
+Consequently, we chose this part for the functional assay, as it proved to be more obvious in detecting the change of colour in ''E. coli'' with the presence of formaldehyde.
-<!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K4813005 SequenceAndFeatures</partinfo>