Part:BBa_K2232020
PsspB-gerAa
This part expresses in B.subsilis to increase the germinability of the spore.
Usage and Biology
This part,PsspB-gerAa (BBa_K2232020),consisting mainly of the PsspB promoter followed by the first 303bp of the gerA gene, plays the role as Responder in the germination of spores. The gerA from Bacillus subtilis encodes the spore’s germinant receptor for L-alanine. Overexpression of this gene will increase markedly the rate of germination of B. subtilis spores even at low L-alanine concentration. The promoter for the B.subtilis sspB gene (PsspB) is stronger than the endogenous gerA promoter, and only switch on the transcription after the initiation of sporulation. Since B.subtilis exhibits accurate and efficient homologous recombination, a single cross-over event between the gerAa region in the BioBrick and the endogenous gerA sequence inserts the PsspB promoter upstream of the gerA cds, which increases the germination rate of Bacillus subtilis endospores eventually.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 7
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 94
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 409
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.
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).
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)
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.
References
- This part created by 2017 SZU-China iGEM team basing on the original part: BBa_K911008
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