Through great amount of RNA-seq and RT-qPCR work, we identified three theanine responsive genes (nasA, amtB, yrbD) in the B.subtilis NCIB 3610. We then fused lacZ reporter gene to nasA, amtB, yrbD respectively.
~How to introduce lacZ sequence?~
lacZ reporter gene was introduced to the target sites via “homologous recombination”.
This year, we have submitted 3 BioBrick parts to the registry. Figure.1 shows their design. In our BioBrick, lacZ and cat (chloramphenicol acetyltransferase) genes are flanked by the DNA sites for recombination. The sequences for recombination are homologous to the sequences flanking the target sites. We named these flanking sequences “recombination site A” and recombination site B”.
When the recombination takes place at these two sites(double recombination), lacZ and cat genes are inserted into the chromosome as illustrated in Figure.2. We designed the recombination sites to insert lacZ immediately downstream of the three theanine responsive genes we had identified.
In our BioBrick parts, lacZ does not include promoter region, so lacZ doesn't have any function by itself unless it is inserted into B.subtilis chromosome. Cat gene can be used as selection marker. The size of recombination sites are about 700 bp each, which is long enough to ensure the stable recombination in B.subtilis. LacZ(without promoter) and cat(with promoter+RBS) gene sequences were amplified by PCR from commercialized plasmid, pMutin2 and pSweet respectively. The sequences of recombination sites were amplified from genomic DNA of B.subtilis with overlap sequences for cloning. Recombination sites, lacZ, cat fragments in our biobrick are assembled together by Gibson assembly.
Illegal restriction sites for iGEM use were later removed by base substitution using gblock Gene Fragments from IDT.
B.subtilis is known to have natural competence of transformation. According to our protocols, our BioBrick parts can be taken into the cells, and can be easily inserted into the chromosome via homologous recombination.
 Dubnau, David. "DNA uptake in bacteria." Annual Reviews in Microbiology 53.1 (1999): 217-244..