Introduction
The first step of every good plan is to have a plan, or to design one. Then you need to build a prototype and test it. When it inevitably fails, you learn from it. This is the engineering cycle that guided us through our project.
Engineering relies heavily on scientific principles to develop systems that address specific problems. Engineering biological systems, which are often intricate and not fully understood, poses unique challenges. Despite the inherent complexities, applying a systematic engineering approach can greatly enhance the overall process.
Figure 1: The engineering success cycle
We had several systems we wanted to develop, and for each of them we used the engineering success cycle approach:
Compete
First Design
We designed the genetic circuit for our project, and appropriate primers, with Benchling. The genetic circuit can be found in the registry - BBa_K4633101.
Figure 2: The general design of our competition genetic circuit, for more information please refer to our compete page or our parts page.
First Build
Step 1: restrict insert and plasmid.
Step 2: ligate.
Step 3: transform into E. coli.
First Test
To confirm the success of our cloning efforts, we conducted colony PCR.
Figure 3: Colony PCR after transformation of FimH-GFP insert. Expected size of 1172bp was achieved in the marked lanes (colonies 6,7,8 and 10). The forward primer was designed to anneal within the inserted FimH sequence, therefore negative colonies should show no amplification.
Second Test
Sent positive colony to sequencing. Received poor quality results - beginning of insert wasn't sequenced.
First Learn
The Benchling primer design tool doesn't check for uniqueness and forward primer annealed in two places, one of which was inside the insert. Decided that moving forward we'll use the SnapGene primer design tool which verifies uniqueness.
Second Design
Selected positive E. coli colonies based on the colony PCR shown above, extracted the plasmid and commenced B. subtilis cloning.
Second Build
Cloning into B. subtilis.
Third Test
We received very few colonies on the transformation plates, the morphology of the colonies was not as described in the literature.
Figure 4: transformation plates of B. subtilis.
Second Learn
In general, it is assumed that if a transformed bacteria grew on plates containing antibiotics, that the resistance is a result of the bacteria receiving a plasmid, and colony PCR is meant to distinguish between bacteria that received a plasmid containing an insert, and a plasmid that self-ligated.
We extracted plasmids from E. coli that we knew contained the insert, due to colony PCR, so all plasmids cloned into B. subtilis should also contain same insert. However, as mentioned and as can be seen in figure 4, the colonies didn't look like B. subtilis.
We performed colony PCR with primers for the plasmid and primers for B. subtilis genome, to confirm if the colonies were B. subtilis and if they did contain our plasmid.
Fourth Test
Run through electroporation gel. Correct size for genomic primers, no amplification for plasmid primers.
Figure 5: Colony PCR results for test with primers for the plasmid. As can be seen, no amplification in any of the lanes.
Figure 6: Colony PCR with genomic primers. As can be seen, amplification in the expected size was observed in several colonies, confirming the bacteria is B. subtilis.
Third Learn
Based on the results, we know that the bacteria that grew on the plates are B. subtilis, but they don't contain our plasmid (we know from the results of the test in E. coli that the primers are functional), since the plasmid was gifted from iGEM Technion 2020, perhaps it was mislabeled and we don't actually have the pBE-S plasmid, but rather a different one.
Fifth Test
Sent plasmid for whole plasmid sequencing.
Fourth Learn and success
Wrong selection: results showed the plasmid had two different antibiotics resistances, one for E. coli and one for B. subtilis. In the next transformations we changed the antibiotics accordingly, and the transformations into B. subtilis were successful.
Protect
First Design
Tried to use paraA as inducible promoter, could not be manufactured.
Second Design
Tried to use paraE as inducible promoter, could not be manufactured.
Third Design
Modified paraE to be used as inducible promoter. For more information on these design iterations, see our protect page.
First Build
Cloned inserts (promoter+RBS+mCherry) into E. coli, for further detail see our parts page.
First Test
Performed colony PCR, found several positive colonies for both inserts.
Figure 7: Colony PCR results for transformations of the killswitch promoters- paraE and paraR (both with mCherry downstream). There was a mistake and a ladder wasn't loaded but it is estimated that the amplification is of the expected size (414bp for paraR and 478bp for paraE). Again, primers were designed that one anneals inside the insert, so with no insert there should be no amplification.
We also saw that paraR colonies turned pink, despite these parts originating from B. subtilis genome and not being intended for E. coli. Since paraR was suspected to be leaky, as detailed in our protect page, we suspected that with the inducer present, paraE might function within E. coli and we will get expression of mCherry from this promoter as well.
Figure 8: Pink E. coli colonies from copy plate.
Fourth Design
Decided to test our systems in E. coli as well.
Second Test
Measured fluorescence in E. coli. Results for paraE were the opposite than expected, lower levels of expression after induction, for more information see our results page.
Figure 9: Normalized fluorescence level from paraE in E. coli.
For paraR it is difficult to determine if the expression levels lowered after the L-arabinose induction or remained the same due to the large error bars.
Figure 10: Normalized fluorescence level from paraR in E. coli.
First Learn and success
We were able to characterize these parts in E. coli. While the parts function, they didn't work as was expected in B. subtilis.
New chassis development
First Design
Extracted growth protocol from literature. For more information please refer to our new chassis development page.
First Build
Executed on the protocol.
First Test
Positive results were obtained, with L. crispatus displaying successful growth.
Figure 11: L. crispatus growth in liquid medium (MRS).
First Learn
While the protocol does work, it is inconvenient, requiring a special "jar" and a new CO2 producing bag whenever the jar is opened (also limited space in jar for plates and tubes).
Figure 12: The anaerobic system used for L. crispatus growth.
Second Design
Modified protocol to simplify it - tried growing tubes and plates outside the jar (and without a CO2 bag). Some plates were wrapped in parafilm to increase anaerobic conditions.
Second Build
Executed modified protocol.
Second Test
Once again, we observed successful growth of L. crispatus.
Figure 13: L. crispatus growth on plate covered with parafilm.
Second Learn and success
L. crispatus growth conditions can be done without special equipment, making the process cheaper and more accessible.