The engineering cycle is made out of four distinct steps; design, build, test and learn. Throughout the cycle, an idea can be turned into a prototype which can be tested. Experience from this test can then be used to inform the next iteration, until a version working as intended is achieved. On this page, we have illustrated the engineering cycle we went through with one part of our project, the creation of new colors of amilCP.

Color mutations - the engineering cycle

Design

In this part of the project, we wanted to find mutations that caused amilCP to take on different colors. We decided to use the E.coli strain DH5-α to express the protein, since they are safe, grow quickly and are well equipped for transformation and thus a good choice for this purpose. To introduce the mutations, we decided to focus on the amino acid position N157 in the protein (not GFP-numbering). We opted to design primers to introduce various specific amino acid substitutions via PCR, as well as design primers with completely random bases at amino acid position N157. Inverse PCR was selected for this purpose since it avoided having to assemble the various parts, thus reducing the complexity of the design and improving its probability of success.

For our backbone, we chose pSB1K3. This was partly because it was convenient as we had access to the gene in this backbone. However, the fact that the plasmid was high-copy suited our experiments, as it made it easier to clearly see if new colors developed. Kanamycin as the selection marker was chosen over chloramphenicol since our control mRFP1 had the same resistance, enabling restreaking them on the same plates.

To test whether the design worked, we chose to firstly visually evaluate the color of the colonies. If they obtained a different color than E.coli that doesn’t express any chromoproteins or other pigments and a different shade than the already discovered blue, green and pink variants of amilCP, we would know that the implementation was successful. In that case, we would move forward with further characterization, with an assay of maturation time, to evaluate the viability of the modified parts, as well as measure the absorbance maximum of the chromoprotein.

The primers were designed in such a way that the forward and reverse primers were positioned right next to each other (Figure 1), which enabled amplification of the whole plasmid through inverse PCR. We designed the primers in SnapGene, and calculated annealing temperatures using Thermo Fisher’s Tm calculator. The difference in melting points in primer pairs, the GC content, probability of the primers forming hairpin loops or self-dimers were all calculated using IDT’s OligoAnalyzer and taken into account when designing the primers.

Figure 1: All primers for the color mutations experiments. F = Forward, R = Reverse. One of two reverse primers were used for each specific mutant, depending on the melting point of the forward primer.

Build

The primers were phosphorylated at the 5’ end to enable ligation of the amplified segment into a circular plasmid after the inverse PCR. To confirm the inverse PCR worked as expected, agarose gel electrophoresis was used. A band around 2800 bp indicated successful amplification (Figure 2). After self-ligation using T4 DNA ligase, the plasmid was then transformed into competent DH5-α E.coli. Since we chose pSB1K3 as the vector we used kanamycin as the selection marker.

Figure 2: Agarose gel (1%) of PCR product after inverse PCR of the color mutations.

To confirm that the build worked as intended, in other words that the mutations were the right ones and at the right position, colony PCR was performed. Primers annealing to both ends of the amilCP gene were used, and the PCR product was analyzed by agarose gel electrophoresis to confirm the size. The PCR products were then sent to Eurofins for Sanger sequencing.

Test

To test and characterize the correctly constructed builds, we measured their maturation times and absorbance maximum. The former is an important value when evaluating the protein’s usefulness, and the latter helps to more precisely and quantitatively characterize its function than the qualitative visual inspection done by eye.

The maturation assay utilized a camera to automatically take pictures of restreak LB-agar plates of the mutants at regular intervals during a 96 hour period (Figure 3). mRFP1 and green amilCP were used as controls on all plates, which helped to evaluate the accuracy of the analysis by comparing the variance between plates.

Figure 3: The plates from the maturation assay.

The final data result attained through this analysis is the chromoproteins halftime value. This is the time it takes for half of the maximum intensity to be reached. More information about these results can be found on AmilCP results.

Learn

Through this engineering cycle, we reaffirmed previous observations that position 157 is important for the color of the chromoprotein. Since the aim of this part of the project was to find new colors of amilCP, we can conclude that our design and the engineering cycle was successful. The new and characterized colors (Figure 4) have been submitted as new parts to the registry, and can be found here.

Figure 4: Some of the colors we got from the introduced mutations.

If we were to do a similar project in the future, we would try to improve the test stage, specifically the maturation assay. The odd behaviors seen in the results from the maturation assay indicate some fine tuning of the setup is necessary for more accurate results. This would include making sure the camera settings are always the same and don’t adjust automatically, and that the lighting is good and constant.