Part:BBa_K4204020
Crystal violet induced GFP improved with ProQC system
Overview
This part is a combination of BBa_K4204019 and BBa_K4204016. It contains both the crystal violet induction system (a sensitive induction system that uses crystal violet as a cheap inducer) and the protein quality control system (a system that increases the integrity of protein by locking the translation of truncated mRNA). This part serves as a universal expression cascade-the GFP in the middle of this part could be replaced by any coding sequence. By using this part, one could use a cheap inducer to achieve high-quality protein expression, which allows the price of the protein product to be lower. This could make their product more affordable for consumers.
Crystal violet induction system
introduction
Crystal violet induction system is a cheap, efficient and sensitive induction system. The repressor protein EilR could bind to operator eilOc and inhibit transcription when crystal violet is absent. Its working principle is shown in the figure below.
Characterization
BBa_K4204016, a crystal violet-induced sfGFP expression system, is used to characterize the crystal violet induction system, in which sfGFP is used as a reporter, and the relative fluorescent level per cell under a certain concentration of crystal violet could represent the expression level of the induction system under that inducer concentration.
We did a kinetics assay to characterize the induction curve of the crystal violet induction system. 8 different concentrations of crystal violet are tested with 6 repeats each. The detailed plate setup is below (Table 1).
The result (shown in Fig.2) shows that the minimal concentration of crystal violet required to get observable protein expression is 0.02um, and the expression level reaches a maximum when crystal violet concentration reaches 0.2um. For higher concentrations of crystal violet, the expression level per strain decreases by about 25% while the exponential phase of strain growth delays, probably due to the burden of expression and the slight cytotoxicity of crystal violet. Overall, the optimal concentration of crystal violet for induction is 0.2um.
Protein quality control system
Introduction
The protein quality control system (ProQC system) is a system that could enhance the integrity of the protein product by preventing the translation of truncated mRNA. It involves a prefix (switch) and a suffix (trigger) on the two sides of the ORF. Our team applied it to LysPBC5 expression and improved upon BBa_K653003 to get BBa_K4204019 as a universal expression model for the ProQC system for future iGEM teams to use (in which the GFP could be replaced by other coding sequences). This page compares the western blotting result with and without the ProQC system to prove its effectiveness. Since we need to extract protein and do western blotting to verify the effect, His-tagged LysPBC5 is used as an example protein for efficiency.
Western blotting result without ProQC system
Fig. 1: WB result without ProQC system. (from left to right: Cas12b protein, LysPBC5 protein, LysPBC5 protein, cell lysate flow through of Cas12b protein)
Although the size of LysPBC5 protein (38Kda) is correct*, several shorter bands also appeared on the image (meaning that they also have his tag), revealing that shorter, nonfunctional versions of the protein exist in the sample. This reflected that a bunch of truncated mRNA might be produced during transcription, which not only wastes energy and amino acids but also traps ribosomes (since truncated mRNA does not contain a stop codon).
"*"The hollow bands in the middle of the lane of protein LysPBC5 are a consequence of the high protein concentration of purified LysPBC5. High protein concentration leads to high primary antibody levels, and thus causes a high concentration of secondary antibodies with Horseradish Peroxidase to stack in the middle of the band, which leads to fast depletion of fluorescence substrates in the center.
Western blotting result with ProQC system
We used protein expressed by BBa_K4204014 to characterize the effectiveness of the ProQC system. The western blotting result of LysPBC5 with the Pro-QC system shows significant improvement in the integrity of the protein. LysPBC5 is diluted four 4 times before being loaded. Rows 7 to 10, which correspond to eluted LysPBC5 with the ProQC system, are clearly visible in the membrane picture obtained using ECL imaging (Fig. 2), demonstrating that the LysPBC5 protein with the ProQC system is being produced appropriately and that few proteins are lost during the washing process (lanes 1-6 that contains wash through are empty or nearly empty). Western blot analysis reveals that only the correct and precise target bands are being expressed, with the mass of protein being around 40kDa (the calculated mass of LysPBC5 is 38 kDa) and no aberrant or interfering stripes in the result. Overall, the WB result with the ProQC system shows that it is useful in improving the full-length expression rate of LysPBC5, which also means that it's applicable to other proteins' expression.
Figure.2 The WB result of LysPBC5 protein with protein quality control system (Lanes 1-6, 7-10, and 11-13 contain wash-through, eluted protein, and cell lysate flow-through, respectively, the gel is stained after transmembrane to show that very few protein left on the gel)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1166
Illegal NheI site found at 1248
Illegal NheI site found at 2332 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1045
Illegal BamHI site found at 1943 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 475
Illegal NgoMIV site found at 1995
Illegal AgeI site found at 625 - 1000COMPATIBLE WITH RFC[1000]
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