Synthetic biology has become one of the promising technologies around the globe. The intersection between biology and engineering aims to construct biological systems for a helpful purpose. The framework of engineering cycles is crucial to succeed since they facilitate the systematic development and optimization of biological systems to execute designated functions. Navigating through our project, we encountered numerous challenges that demanded innovative solutions and a rethinking of our designs. Here, we illustrate our iterations within the engineering design cycles.
We decided to regulate the operon via a negative feedback loop dependent on the terephthalic acid concentration. XylS/pS1/pS2 expression system is a naturally occurring expression system that we wanted to use.
The pS1/pS2 expression system was exchanged with the constitutive promoter pEM7 with a Shine-Dalgarno sequence to increase basal XylS-mt expression. mKate2 fluorescence read-out overall was higher compared to the initial system. However, the expression was less sensitive to TPA induction.
As we needed the gene for AlkS, we wanted to order the fragment.
Due to the high complexity of the fragment, a synthesis was not possible. We learned that the size of a construct matters.
We order it as three fragments and one backbone. We assembled the three fragments without the backbone, PCR amplified, and purified. The fragment sequence was tested with an analytical agarose gel and sequencing. The single AlkS fragment and backbone were then successfully added.
To improve the solubility and uptake of long-chain alkanes, we used a biosurfactant. (Chen et al., 2023) used rhamnolipids to successfully suspend the alkanes and induce the AlkS/pAlkB expression system in E. coli.
The alkanes were resuspended in varying rhamnolipid concentrations. Cell growth and fluorescence was measured over a few hour period. No usable results were generated due to the red coloring of the rhamnolipids, which masked the fluorescence emitted by mKate2. (Chen et al., 2023) worked in E. coli with the fluorescent reporter protein GFP and did not have an issue with overlapping color.
Two different biosurfactants, Tween® 80 and DMSO, were tested to suspend the alkanes and proper fluorescence measurements were taken.
Having settled for the idea of creating a digital twin for our organism, we researched existing models for P. fluorescens and found one that promised a high accuracy. Using this model we wanted to predict how well the colony would grow in specific conditions and when the resources would be depleted.
We ran different simulations and found out that using this model, the results only indicate how much the colony grows at one point, when the initial medium concentration is present.
As we needed to know the proportions of the medium and colony at all times, this steady-state modeling was not suitable for us. By using dynamic flux balance analysis, this problem was solved later on.
Having established the dynamic flux balance analysis, we attempted a second round of calculating the growth and medium composition.
This time, the analysis produced results where the colony starts small and grows over time, while consuming energy from the medium. However, comparing the in silico results to the experimentally measured data showed significant differences.
By applying a correction factor k = 0.04 the model produced reliable results over multiple iterations during the dFBA.
Chen, D., Xu, S., Li, S., Tao, S., Li, L., Chen, S., & Wu, L. (2023). Directly Evolved AlkS-Based Biosensor Platform for Monitoring and High-Throughput Screening of Alkane Production. ACS synthetic biology, 12(3), 832–841. https://doi.org/10.1021/acssynbio.2c00620
González-Pérez, M. M., Ramos, J. L., & Marqués, S. (2004). Cellular XylS levels are a function of transcription of xylS from two independent promoters and the differential efficiency of translation of the two mRNAs. Journal of bacteriology, 186(6), 1898–1901. https://doi.org/10.1128/JB.186.6.1898-1901.2003