Part:BBa_K3892016
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 75
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
J23110-G10 leader-EforRed
The BioBrick BBa_K1073022, a chromoprotein called EforRed with a strong promoter (BBa_J23100) and a weak RBS region (BBa_B0032), was improved as a part of our project. In this improved version, the weak RBS region was replaced with a stronger G-10 leader RBS (from BBa_K1758100). The G-10 leader contains longer spacer regions before and after the RBS, leading to a more efficient ribosome binding, and thus, more protein production [1]. In both the old and improved part, a glycine was added after the methionine in order to include a BamHI restriction enzyme site. This was done in order to enable the swapping of the promoter and RBS region. EforRed is fluorescent with an excitation maximum at 589 nm and an emission maximum at 609 nm [2]. It has a bright pink color under white light. Due to the bright color and fluorescence, EforRed can be used as an alternative for the commonly used blue-white screening. This improvement showed a quicker color development on agar plate colonies while exhibiting a higher ceiling of fluorescence in liquid cultures.
Expression
Both BioBricks were expressed in Escherichia coli (E. coli, strain BL21 DE3) and in Vibrio natriegens (V. natriegens, strain Vmax). The BioBricks were transformed into both bacterias in a pUC19 plasmid. Bacteria were grown on LB-Miller agar plates (with V2 salts for V. natriegens) with carbenicillin (100 µg/mL for E. coli, 250 µg/mL for V. natriegens) until red colonies could be seen (16 h). The fluorescence intensity of single colonies was measured using an epifluorescence microscope (Leica Spectraview). To excite the EforRed chromophore, light at a wavelength of 535 nm was used with an exposure time of 50 msec. Fluorescence between 400 and 700 nm was measured. Five similarly sized colonies from one plate of both the improved and the old version of the BioBrick were measured. A single colony was exposed to the fluorescence and an image was taken immediately in order to avoid bleaching of the color (Figure 2). The fluorescence was measured at the brightest part of each image (Green dot in FIgure 2, 3D). In E. coli (BL21 DE3) the improved part had 40% higher fluorescence and in Vmax, the fluorescence was over twice as high in the new part, although colonies exhibited a wide range of EforRed fluorescence (Figure 3).
Fluorescence was also measured from liquid cultures using a plate reader (BMG Clariostar). Liquid cultures of V. natriegens (Vmax) and E. coli (BL21 DE3) with both the old and improved BioBrick were made. The flasks (Erlenmeyer) had an open atmosphere (cotton plugs) and liquid media (LB-Miller 100 µg/mL carbenicillin for E. coli and V2 salts and 600 µg/mL carbenicillin for V. natriegens, 10 mL volume) at 1/10th of the flask volume. In addition, liquid cultures of V. natriegens (Vmax) in baffled flasks were also used in this experiment. To excite the EforRed chromophore, light at a wavelength of 554 nm was used. Fluorescence was then measured at 586 nm. Optical density was measured at 600 nm. Four biological samples from each test group were measured after 24 h and 96 h incubation at 37 °C. The relative intensity of each sample was divided by the optical density (600 nm) to accurately show the protein expression even if the cultures were not equally dense. After 96 h incubation, the fluorescence of the improved part is noticeably higher in all test cultures (Figure 3).
| None |