Revision as of 09:26, 11 October 2023

pETM6-pnar-mCherry

pETM6-pnar-mCherry

pETM6_pnar-mCherry - BBa K4842003

Construction Design

The Part consists of three parts: vector backbone pETM6 (BBa_K4842000 Part:BBa K4842000 - parts.igem.org), gene fragment pnar (promoter/BBa_K4842001 Part:BBa K4842001 - parts.igem.org), and fluorescent tag mCherry (reporter/BBa_J04450 Part:BBa J04450 - parts.igem.org).

Figure 1 shows the construction design:

Figure 1: Construction result of the plasmid pETM6-pnar.

Engineering Principle

Expression of soybean glycoside conversion to (S)-estradiol biosynthesis pathway genes (dznr, ddrc, dhdr, and thdr) in engineered Escherichia coli was controlled by an anaerobically-induced nar promoter[1], which both avoids the use of the costly inducer ITPG during the fermentation process and provides a microsoluble oxygen environment that is conducive to (S)-estradiol biosynthesis[2]. And to facilitate the study, we utilized the fluorescent tag mCherry for screening in the preliminary stage of the experiment[3]. We hypothesized that the expression of the fluorescent tag mCherry could characterize the expression of (S)-estradiol biosynthesis pathway genes (dznr, ddrc, dhdr, and thdr) to a certain extent.

Figure 2 illustrates the engineering principle:

Figure 2: Engineering principle of controlling gene expression using the nar promoter.

Experimental Approach

1. Construction of plasmid pETM6-pnar

To construct the plasmid pETM6-pnar, we first double-digested the synthetic pCDM4-pnar and pETM6 with NdeI and AvrII restriction enzymes, respectively. After recycling the target fragments, we ligated the nar fragment with the pETM6 vector using T4 DNA ligase to obtain the complete recombinant plasmid.

Figure 3 displays the construction result of the plasmid pETM6-pnar:

Figure 3: Construction result of the plasmid pETM6-pnar.

2. Construction of plasmid pETM6-pnar-mCherry

To construct plasmid pETM6-pnar-mCherry, we first double-digested pET28a-mCherry and pETM6-pnar with XhoI and AvrII restriction enzymes, respectively. After recycling the target fragments, we ligated the mCherry fragment with the pETM6-pnar vector using T4 DNA ligase to obtain the complete recombinant plasmid.

Figure 4 displays the construction result of the plasmid pETM6-pnar-mCherry:

Figure 4: Construction result of the plasmid pETM6-pnar-mCherry.

3. Mutation library construction for the plasmids pETM6-pnar-RBSx-mCherry

We designed eight RBS sequences downstream of the nar promoter using the RBS Calculator (https://salislab.net/software/), as shown in the table below.

Table 1: RBS sequences designed by RBS Calculator.

We then introduced the RBS sequence mutations by whole plasmid PCR. As shown in Figure 5, we successfully amplified pETM6-pnar-RBS(1-8)-mCherry plasmids containing different RBS sequences.

Figure 5: Electrophoresis results of whole plasmid PCR.

We transformed pETM6-pnar-RBS (1-8)-mCherry plasmids into competent E. coli BL21(DE3) cells, respectively. After overnight culture, positive transformants grew on LB plates. Colony PCR identification showed the correct bands for pETM6-pnar-RBS (1-5)-mCherry transformants, while no bands were observed for pETM6-pnar-RBS (6-8)-mCherry. This may be due to mistakes in picking colonies or omissions in adding templates. Nevertheless, we inoculated all eight transformants for subsequent fluorescence intensity testing.

Figure 6: Transformation and colony PCR identification results of plasmids pETM6-pnar-RBSx-mCherry.

Characterization/Measurement

4. Fluorescence intensity detection of RBS mutant library.

We measured mCherry expression every half hour after induction using a fluorometer. Cell density (OD600) was measured on a UV/VIS spectrophotometer. The total red fluorescence of whole cells was determined by the fluorometer with excitation at 580 nm and emission at 610 nm. Background from transformants with mutant promoter plasmids was subtracted under the same conditions to obtain actual red fluorescence intensity.

Figure 7 shows the fluorescence intensity assay results of the RBS mutant library:

Figure 7: Fluorescence intensity assay results of the RBS mutant library.

In summary, using mCherry as a model protein, we successfully constructed the pETM6-pnar-mCherry plasmid. Based on this, we obtained a nar promoter library with different RBS sequences. The mutant library was characterized by fluorescence intensity detection, and the three best-performing RBS sequences were identified. These RBS sequences have the potential to achieve high-level regulated expression of (S)-equol pathway enzymes.

Reference

[1] Lee P G, Kim J, Kim E J, et al. P212A mutant of dihydrodaidzein reductase enhances (S)-equol production and enantioselectivity in a recombinant Escherichia coli whole-cell reaction system[J]. Applied and Environmental Microbiology, 2016, 82(7): 1992-2002.

[2] Clomburg J M, Blankschien M D, Vick J E, et al. Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids[J]. Metab Eng, 2015, 28(202-212).

[3] Lee P G, Lee S H, Kim J, et al. Polymeric solvent engineering for gram/liter scale production of a water-insoluble isoflavone derivative, (S)-equol[J]. Applied microbiology and biotechnology, 2018, 102: 6915-6921.

Sequence and Features