Expression of mini-sMMO in B. subtilis
Our DNA construct (part BBa_K5128100) is based on the plasmid pDR110 and mini-sMMO gene fragments. The amyE front and amyE back sections enable mini-sMMO to be inserted into the middle of the B. subtilis amylase (amyE) gene via homologous recombination. The spectinomycin resistance gene specR enables us to screen for transformed colonies. Since methanol, the product of mini-sMMO acting on methane, is toxic to B. subtilis, we also included the lacI operon, so that mini-sMMO will be expressed only when IPTG is added.
We ordered mini-SMMO gene fragments from IDT, but IDT was unable to synthesize the gene fragment for the alpha subunit. Subsequently, we re-designed the alpha fragment and split it into two parts, which we then re-ordered. Using overlap-extension PCR, we stitched together these two parts into the alpha fragment.
The left lane is the New England Biolabs 1 kb Plus DNA Ladder, and the right lane is the overlap-extension PCR product. The bright band in the right lane is consistent with 2600 bp, the length of our alpha fragment.
We assembled our four fragments (amyE back, alpha fragment, beta fragment, amyE front) using NEBuilder HiFi DNA Assembly (similar to Gibson assembly). This was then transformed into B. subtilis strain 168 and streaked on LB/agar/spec plates to grow at 37°C overnight. This first transformation attempt was unsuccessful, as we ended up with no colonies on our plates.
For our second transformation attempt, we skipped the overlap-extension PCR and used the NEBuilder HIFi Assembly to directly assemble the five fragments (amyE back, alpha_1 fragment, alpha_2 fragment, beta fragment, amyE front). We also ran our PCR in triplicate to ensure that we had plenty of starting material to work with, and we added a quality control step by verifying our PCR products on a gel.
From right to left: ladder (New England Biolabs 1 kb Plus DNA Ladder), amyE back, apha_1, alpha_2, beta, amyE front, and then the 5 fragments are repeated in order. Each of the lanes have clear bands at the right position.
Following assembly, the product was transformed into B. subtilis strain 168 and streaked on LB/agar/spec plates to grow at 37°C overnight. After the first night, we saw three colonies. After the second night, we saw many more colonies, including on the negative control plates, so we decided to move ahead with just the three colonies from the first night incubation. We re-streaked each of the three colonies on LB/agar/spec plates and on LB/starch plates.
Following incubation at 37°C overnight, we saw growth on all the plates. On the LB/starch plates, we performed the iodine-starch test. Starch and iodine combine to make a dark purplish color, and a yellowish halo around a colony indicates a deficit of starch around the colony, suggesting that the amylase gene (amyE) is present. Conversely, a lack of a halo suggests that amyE is absent and that the colony has been successfully transformed.
AmyE+ indicates that the amylase gene is still present, whereas, AmyE- indicates that the amylase gene is not present, suggesting a successful transformation.
We interpret the starch test as showing that one of the colonies is amyE negative. Going forward, we plan to do further experiments to check if that colony has truly been successfully transformed.
Thermocycler
Our team developed a Peltier-module-based PCR machine that can be built for half the price of the cheapest available commercial offering. The design and code is shared on GitHub. Despite the shortcomings of our design, such as the lack of a heated lid and its unwieldiness, we were able to perform a successful test run during our “Get Ready for Synthetic Biology” class that we taught to middle school students. In the future, we hope to update the PCB design to significantly reduce the amount of wiring and work to select more temperature resistant Peltier modules.
An image of the thermocycler
A gel that we ran on the PCR product from our thermocycler
Microcentrifuge
We successfully developed a microcentrifuge rotor designed to fit on top of a standard 1000kv drone motor. The design is shared on Thingiverse. This brought the cost of the microcentrifuge down to $35, including the cost of 3D printing, which is less than half the price of the cheapest online model. By filming a slow-motion video of the centrifuge in action, we estimated the rpm of the microcentrifuge and calculated an RCF of 5000g. This value, while not anywhere close to more expensive lab grade centrifuges, was quite close to the cheapest online model that we found. In the future, we hope to design an enclosure for the centrifuge to increase safety.
An image of the microcentrifuge