Part:BBa_K2232020:Experience

iGEM2017 SZU-China

The part was synthesized and insert into the expression vector by restriction sites BamHI and HindIII（Fig.1）, and the correct construction of this recombinant plasmid was confirmed by PCR identification and sequencing of the PCR products.

Fig.1 Construction of the expression vector_PsspB-gerAa. The Rep_B and Kan represents replicon of B.subtilis and kanamycin resistance marker. The part PsspB-gerAa was inserted by the restriction site BamHI at 4151 bp and HindIII at 4652 bp.

We transformed the expression vectors into Bacillus subtilis WB800 by optimization of Spizizen method, and the real positive clones was confirmed by kanamycin screening and nucleic acid electrophoresis (Fig.2).

Fig.2 1% Agarose Gel Electrophoresis of DNA extracted from the positive clones and its identification by restriction digestion. The product of plasmid digested showed two signal bands at 501 bp and 6393 bp respectively, which correspond to the length of PsspB-gerAa and the blank plasmid. Lane 1: Complete plasmid; Lane 2: Plasmid digested by BamHI and HindIII; Lane M: DL marker.

In order to test the germination rate of the B.subtilis endospores, we used the whole yeast solid plate (Fig.3) and the 2xSG liquid medium (Fig.4) to harvest the mature spores, The yield of spores can be calculate by phase contrast microscope using the Hemocytometer，which always reach 90% in 72h after cultivating（Fig.5）

Fig.3 The whole yeast solid plate.

Fig.4 2xSG liquid medium.

Fig.5 The endospores of B.subtilis observed under the phase contrast microscope. Sporation rate always reach 90% above in 72h after cultivating at the whole yeast solid plate and 2xSG liquid medium.

To verify the increases in the germination rates with L-alanine of spores transformed this part PsspB-gerAa (BBa_K2232020), both spores of original strain (control group) and transformed strain (gerAa group) were collected and detected the germination rates induced by L-alanine(2mmol/L). Germination was monitored by reading the drop in absorbance (A600) in a 96-well microplate reader (Molecular Devices FlexStation 3).Theoretically, the earliest easily measured event in spore germination is the release of various ions, and Ca²⁺-DPA release is accompanied by the loss of 70% of the total amount of the OD600 that is lost upon spore germination. Consequently, measurement of the OD600 of germinating spores, in particular, the maximum rate of the fall of the OD600 at a given germinant concentration, is a simple and reliable method for quantitating and comparing rates of spore germination. As shown in Fig.6 and Fig.7, the gerAa group showed a higher proportion in germination and quicker rate in germination than the control group, which indicated the gerA play a good Responder in germination to L-analine in particular at low concentration. Therefore, gene gerAa is verified to be in good condition and can work efficiently as repected.

Fig.6 Variation of values and rates of loss in OD600 during germination of spores with or without transformation of PsspB-gerAa. The results represent the average (SD) of three independent spore batches The drop rate of OD600 is proportional to the germination rate and the value of gerAa group is more than twice of control group with significant difference in statistics.

Fig.7 Variation of values and rates of loss in OD600 during germination of spores with or without transformation of PsspB-gerAa. The results represent the average (SD) of three independent spore batches The drop rate of OD600 is proportional to the germination rate and the value of gerAa group is more than twice of control group with significant difference in statistics.

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