Difference between revisions of "Part:BBa K3739085"

Revision as of 23:33, 21 October 2021

K525998(T7-RBS)-LMT-his-hutH-FT

Histidine ammonia-lyase with a his-tag is involved in catalyzing the reaction of changing L-histidine into trans-urocanate. LMT is a signal peptile. The proteins labeled of tetracysteine motif tag (FlAsH tag, FT in short) can be detected with the biarsenical compound FlAsH.

Biology

FlAsH system was previously reported as a fluorescence detector for secreted proteins1. The protein labeled of tetracysteine motif tag (FlAsH tag, FT in short) can react with biarsenical compound FlAsH, showing great fluorescence at 528 nm emission. FlAsH system is used to quantify and qualify the secretion efficiency. LMT is a signal peptide, and we used LMT-his-hutH-FT to verify the secretion effect of LMT.

Usage

We ligased the induced promoter+RBS (BBa_K525998) and the parts (BBa_K3739083) on the expression vector pET-28a(+) by standard assembly (BBa_K3739085). Then the ligation mixture was transformed into E.coli BL21 (DE3), and the correct recombinant one was confirmed by kanamycin, colony PCR and sequencing.

Characterization

1. Agarose Gel Electrophoresis

After BBa_K3739085 was constructed on vector pET-28a(+) and transformed into E.coli BL21 (DE3), colony PCR was done to verify that the plasmid was correct. Data is shown below:

Fig. 1. Colony PCR results of BBa_K3739085

2. Qualification of LMT secretion efficiency

After cultivation for 5 hours at 37℃ and induction for 2 hours with 0.1mM IPTG, the cell supernatant was separated by centrifugation. The supernatant along with 2 μM FlAsH-EDT2 and 1 mM DTT were added to wells in a 96-well plate. Following incubation in the dark for 1 h at 37 °C, fluorescence was measured by 503 nm excitation and 528 nm emission, and each value was normalized by OD₆₀₀.

Fig. 2. Fluorescence intensity/OD₆₀₀ of cell supernatant after incubation in the dark for 1h.
The result shows that the fluorescence intensity/OD₆₀₀ of the group Aly01 and LMT is significantly higher than native control group, which proves that these 2 signal peptides can function well.

3.dynamic model of recombinant protein secretion

To promote our understanding on secretion process mediated by signal peptides, we built a model about relationship between the amount of secreted recombinant protein and time. In order to get the value of P_T (the total amount of recombinant protein per unit volume of reactor), P_M (the amount of the recombinant protein in the medium compartment in unit volume) andγ(extracellular secretion rate constant), a sets of characterization experiments were done.
BBa_K3739087 were constructed on plasmid backbone pET-28a(+) and then the recombinant plasmid was transformed into E. coli BL21 (DE3). After cultivation for 5 hours and induction for 3 hours with 0.1mM IPTG, 50mg/L chloroamphenicol was added to bog down the synthesis of protein in the engineered bacteria in order to make the experimental operations more convenient. Then, the cell supernatant was collected in 0, 6, 12, 18, 24, 30, 40 min to test the amount of secreted recombinant protein by fluorescence detection of FlAsH-EDT2 and FT.

Fig. 3. the fitting of equations and experiments results.
The data shows that after the protein synthesis was inhibited, the change in the amount of secreted protein over time was well matched with the model.

Reference

1. Haitjema, C. H.; Boock, J. T.; Natarajan, A.; Dominguez, M. A.; Gardner, J. G.; Keating, D. H.; Withers, S. T.; DeLisa, M. P., Universal Genetic Assay for Engineering Extracellular Protein Expression. ACS Synthetic Biology 2014, 3 (2), 74-82

Sequence and Features