Part:BBa_K5398605

Properties: This plasmid contains all the basic genetic parts required for the bacterial expression of Mfp6 protein(BBa_K5398601)</b> with the small ubiquitin-like modifier (SUMO) protein(BBa_K4613016). Finally, the Mfp6 protein(BBa_K5398601)</b> was successfully expressed in E. coli BL21(DE3) cells containing this plasmid.

Usage and Biology

Mussels foot protein type 6 (Mfp6)(BBa_K5398601) is a 13.8 kDa protein in mussel byssal gland cells, essential for maintaining the reducing conditions needed for optimal wet adhesion. It's rich in cysteine, which forms stable structures and provides antioxidant protection, preventing over-oxidation of dopamine residues in adhesion proteins like Mfp5, thus preserving their adhesive function. Mfp6(BBa_K5398601) also potentially regulates the tautomeric balance of these proteins, influencing their adhesion performance.

This genetic device is a improtant part of the Tyrosinase Catalysis System.When tyrosinase TyrVs(BBa_K5398600) causes excessive oxidation of tyrosine on the fusion protein TRn4-Mfp5(BBa_K5398020) to form dopaquinone, the Mfp6 protein(BBa_K5398601) can reduce the excessively oxidized dopaquinone back to dopamine, thereby enhancing the adhesive performance of the fusion protein TRn4-Mfp5(BBa_K5398020).

Fig. 1 Mechanism of action.

Basic parts assembled for device construction

The part sample which is flanked at the beginning and the end with prefix and suffix respectively, is composed of the following basic parts assembled together in series and downstream of the prefix:

Bacterial terminator for LacI CDS (upstream of prefix): Putative bacterial transcription terminator

Biobrick Prefix sequence: BioBrick prefix for parts that do not start with "ATG"

LacI Coding sequence: Lac repressor

LacI promoter: Promoter that has the transcriptional control of Lac repressor

T7 promoter: promoter for bacteriophage T7 RNA polymerase

Lac operator: Lac repressor protein binding site

Ribosome binding site: CAT-Seq Ribosome Binding Site

6×His tags: 6×His affinity tag

Thrombin site: Thrombin recognition and cleavage site

T7 tag: T7 tag protein

8×His tags: 8×His affinity tag

SUMO tag: Small Ubiquitin-like Modifier

TEV site: Tobacco Etch Virus (TEV) protease cleavage site

Mfp6 CDS: Mfp6 protein derived from mussel byssal gland cells

Biobrick Suffix sequence : universal suffix for all parts

T7 terminator (downstream of Suffix): terminator for bacteriophage T7 RNA polymerase

Fig 2. The plasmid map of pET28a(+)-Mfp6

Cloning strategy and results

Plasmid Construction

The mfp6(BBa_K5398601) sequence(363 bp) was cloned from the pETDuet-1-Mfp6 vector using a combined gradient and touchdown polymerase chain reaction(PCR) method. Specific primers were synthesized for PCR amplification(Table 1). The forward primer was designated as Mfp6-top, and the reverse primer as Mfp6-bottom. These primers, along with 2×Phanta Max Master Mix (Dye Plus), were used in a touchdown PCR reaction for 30 cycles with a temperature profile of 15 seconds at 95°C, 15 seconds at 56°C, and 1 minute at 72°C.

Similarly, the pET28a(+) sequence(5725 bp) was cloned from the pET28a(+)-TRn4-Mfp5 vector. The forward primer was pET28a(+)-top and the reverse primer was pET28a(+)-bottom(Table 1). The touchdown PCR reaction was performed for 30 cycles with a temperature profile of 95°C for 15 seconds, 67°C for 15 seconds, and 72°C for 1 minute.

Table 1. Sequence of Primer

The amplification products were analyzed by electrophoresis on 1% agarose gels stained with ethidium bromide(Fig. 3). An approximately 300 bp-specific Mfp(BBa_K5398601) PCR product was inserted into a pCR pET28a(+) vector for sequencing using the ClonExpress Ⅱ One Step Cloning Kit.

Fig. 3 1 % agarose gel electrophoresis of the PCR amplified Mfp6 and pET28a(+) vector. Line 1:5000bp DNA Marker. Lines 2-3: the PCR amplified Mfp6(363 bp). Lines 4-5: the PCR amplified pET28a(+) vector(5725 bp).

Transformation and Colony PCR

The final products named pET28a(+)-Mfp6(BBa_K5398605) assembly underwent transformation into E.coli DH5a competent cells and then colony PCR was performed, using T7 and Mfp6 bottom primers(Table 1). For the colony PCR procedure, from the agar plate half amount of each colony was picked and diluted on 10 μl of doble distilled wate. 1 μL was used for sample preparation, while the remainder was used for liquid culture. The samples were loaded and run in 1% agarose gel electrophoresis and then we concluded that the recombination was successful(Fig. 4).

Fig. 4 Colony PCR of E-coli DH5a transformants using T7 and Mfp6-bottom primers. Line: 2000 bp DNA Marker. Lines 2-9: pET28a(+)-Mfp6 using T7 and Mfp6-bottom primers (912 bp) from different colonies.

Sequencing

We selected colonies from Line 3 and Line 4 and performed overnight cultures in tubes containing 5 ml of medium. Subsequently, we extracted the plasmids using the FastPure Plasmid Mini kit and submitted them for sequencing. The sequencing results further confirmed the success of our recombination experiment(Fig. 5).

Fig. 5 Result of pET28a(+)-Mfp6 sequencing.

Reference

[1] Nicklisch SC, Das S, Martinez Rodriguez NR, et al. Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein-6[J]. Biotechnol Prog, 2013, 29(6):1587-1593.