We hope our basic and composite parts are useful for iGEM teams in the future looking to work with B. subtilis, metabolic pathways, and/or potentially expanding on our work of developing a probiotic therapy for Maple Syrup Urine Disease.

Constructing Parts

Compared to E. coli, transforming plasmids into B. subtilis is a more complex process that involves concatenating the plasmid into multimers or using linearized DNA. However, once the DNA is inside the cell, integration of the DNA into the genome proceeds automatically via homologous recombination as long as the sequence is flanked by homology arms of sufficient length.

We identified 4 different loci that we could insert our genes into:

1. amyE
2. thrC
3. bkd - the region around the promoter of the bkd operon
4. azl - the region around azlC and azlD, BCAA exporters

AmyE and thrC are commonly used integration loci in B. subtilis, and encode a starch hydrolyzing amylase and threonine synthase respectively (source). In order to screen for successful integration, we can use a starch or threonine auxotrophy test to tell if the amyE or thrC genes were disrupted (more info about the starch test can be found on Experiments.)

The following genes were integrated into each loci:

1. Extra copies of bcaP and ilvE/ilvK into amyE
2. Pveg into bkd to replace the native bkd operon promoter
3. Spectinomycin resistance marker into azl to knockout the azlC and azlD BCAA exporters
4. Cre into thrC for recombinase expression

Workflow

We used Golden Gate Assembly following the MoClo assembly standard to assemble most of our plasmids (protocol on Experiments page.) We then transformed the plasmids into NEB 5 alpha E. coli competent cells, screened for white colonies via blue white colony screening, and miniprepped the plasmids out of the cells for assembly into higher level composite parts or transformation into B. subtilis. Along the way, we used various methods such as colony PCR and restriction digests to verify our results. (see more on Experimentspage.

Basic Parts: BBa_K4074000 through BBa_K4074023, and BBa_K4074076 Our Basic parts were synthesized by IDT or Twist, flanked by TypeIIS restriction sites for BbsI and BsaI. We first cloned our Basic parts into pL0 backbones (BBa_K4074066 through BBa_K4074069, kindly provided to us by the Weiss Lab) with Spec resistance using BbsI. Correctly inserting our part would result in the LacZ gene being excised--colonies with successful constructs would be white, while those transformed with the pL0 backbone alone would be blue.

Composite Parts: BBa_K4074024 through BBa_K4074041 and BBa_K4074050 to BBa_K4074065, and BBa_K4074077 We next assembled our pL0’s into pL1’s using pL1 backbones (BBa_K4074070 through BBa_K4074072, once again kindly provided to us by the Weiss Lab) with Amp resistance using BsaI. Blue-white colony screening was once again used in this stage. Each pL1 consists of a promoter, RBS, coding sequence, and terminator.

Finally, we assembled multiple pL1’s together into pL2’s along with a special linking sequence called a pELE (BBa_K4074073 through BBa_K4074075) so we could express multiple genes at one locus in B. subtilis. However, it is crucial that these pL2 backbones have homology regions specific to each locus. As a result, we needed to design our own pL2 backbones for each of our loci. To do this, we used the pSB1C3 backbone from the iGEM distribution kit and synthesized inserts with our homology arms through IDT.

Each insert includes the following:

1. LacZ insert for blue-white screening
2. Flanking I-SceI cut sites to linearize our construct for B. subtilis transformation
3. Flanking AatII and NotI cut sites to assemble these inserts into PSB1C3

We successfully assembled these backbones through both traditional assembly and Gibson assembly. Both sets of backbones can be found in the Part Registry.

Using Parts in B. subtilis: After we verified our pL2 constructs, we then needed to linearize the plasmid in order to transform B. subtilis. While we originally intended to accomplish this by cutting the plasmid with I-SceI, we found that we achieved better transformation efficiency by performing PCR on the pL2 plasmid with primers that surround the homology regions.