Engineering Success

“Failure is simply the opportunity to begin again, this time more intelligently.” - Henry Ford


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.

Engineering Cycle

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:


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.

General competition circuit

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.

colony PCR FimH-GFP

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.

B. Subtilis transformation plate 1 B. Subtilis transformation plate 2 B. Subtilis transformation plate 3

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.

colony PCR B. subtilis 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.

colony PCR B. subtilis genome primers

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.


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.

colony PCR E. coli parR paraE

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.

pink E. coli

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.

E. coli paraE expression

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.

E. coli paraR expression

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.

liquid medium

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).

anaerobic jar

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.

parafilm plate

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.