The 2022 UT Austin iGEM team has made it a priority to contribute to the synthetic biology community and pave the way for future iGEM teams and synthetic biologists to conduct high-fidelity research. Among the most notable of our numerous contributions was our involvement in the 2022 Interlab Study through our participation in “Experiment Three” — this experiment focused on plate reader culturing and green fluorescence development over time. Other noteworthy contributions include the identification and deletion of specific genes to improve the potential of engineering Acinetobacter baylyi ADP1 as a chassis organism in synthetic biology. Genetic modifications we conferred on ADP1 were intended to increase the organism’s susceptibility to β-lactam antibiotics and improve homologous recombination efficiency for smaller DNA fragments. Furthermore, we comprehensively describe ADP1 genetic engineering protocols and designs for creating ADP1-based biosensors to detect any DNA sequence of interest. See our Engineering Page for more details on ADP1 genetic modifications and our Experiments Page for important ADP1 protocols. Lastly, our easily adjustable modular detection apparatus is designed as a system that can be easily altered by future iGEM teams and synthetic biologists to detect the presence of DNA sequences.
First, we contributed to the 2022 Interlab study through our participation in “Experiment Three.” In this experiment, we measured how the green fluorescence and optical density of six different genetic devices developed over a six hour period. Each device was cultured in a 96-well plate and a standard culture tube. The 96-well plate protocol was developed by the iGEM Interlab team to be compatible with new automation technology, and the goal of this experiment was to compare the performance of this protocol with that of a standard culture tube protocol. Our team executed this same experiment providing the iGEM Interlab team with data that will be useful in further developing this automation protocol.
We knocked out the pbpG, acrB, and recJ genes from ADP1, with the aim of maximizing ADP1's functionality as a suitable biosensor. By deleting pbpG and acrB from ADP1's genome, we decreased ADP1’s intrinsic resistance to the β-lactam family of antibiotics and increased ADP1's susceptibility to such antibiotics [1]. These genetic modifications provide stronger selection capabilities for ADP1 in the lab. We also deleted recJ to improve ADP1’s ability to recognize and uptake smaller DNA fragments from the environment. Although we were unable to show experimental data to verify the advantage from deleting the recJ gene, Overballe-Petersen et al. shows that knocking out recJ improves the transformation frequency of donor DNA fragments smaller than 1 kb [2]. Alongisde ADP1's natural competence, deleting these genes strengthens ADP1's viability as a chassis organism for future iGEM teams trying to create biosensor systems to detect DNA sequences of interest.
Each DNA sequence and laboratory protocol we used throughout this project is clearly documented on either the parts registry or our wiki. Our wiki and designed parts serve as clear instructions for genetically engineering ADP1. These protocols were designed and documented with the prescience to promote the usage of ADP1 by future iGEM teams and the greater synthetic biology community.
Our team’s most remarkable procedure involves a comprehensive protocol outlining the transformation procedure using ADP1's advantageous natural competence. This complete procedure, documenting how to genetically engineer ADP1, can be located on our Experiments Page under “ADP1 Golden Transformation”. These protocols were designed to be easily implemented by future iGEM teams using ADP1 as a chassis organism. Our ultimate goal is to contribute to the streamlined usage of ADP1 as a chassis organism in synthetic biology. Our Engineering Page provides clear documentation on how to genetically engineer ADP1. Furthermore, our Parts Page includes important DNA sequences, including the tdk/kan selection cassette, which is used to easily confirm successful ADP1 transformants. We hope that these documented ADP1 protocols will encourage future iGEM teams to pursue ADP1-based projects.
In addition to our modular detector sequence, we use the tdk/kan selection cassette in our two-step ADP1 Genetic Engineering protocol. The tdk/kan selection cassette is critical for efficient and accurate ADP1 transformation and DNA detection [4].
The tdk/kan selection cassette consists of a kanR gene, conferring Kanamycin (Kan) resistance, and a tdk gene, conferring Azidothymidine (AZT) susceptibility. Using this tdk/kan cassette allows us to easily select for transformants that have integrated a target DNA sequence. The first transformation step integrates the tdk/kan cassette into ADP1. The second transformation step integrates a "rescue" cassette into the same ADP1 chromosomal location, knocking out the tdk/kan cassette. Growth in the final step indicates that ADP1 has successfully taken up the target DNA sequence. This procedure demonstrates how ADP1 can detect a target DNA sequence. See our Engineering Page and Parts Page for in-depth details on our two-step ADP1 Genetic Engineering Protocol.
The 2022 UT Austin iGEM Team’s Parts Page provides a number of DNA sequences and procedures for genetically engineering Acinetobacter baylyi ADP1. We were able to effectively engineer ADP1's genome using a two-step genetic engineering protocol. See the Engineering Page for more details on how we modified ADP1's genome. On our Parts Page, we explain how our part collection can be used alongside this two-step protocol to delete ADP1 genes, insert DNA sequences into any chromosomal location, and engineer an ADP1-based biosensor to detect any DNA sequence of interest. We created this part collection to guide future iGEM teams in engineering ADP1 and utilizing ADP1’s flexibility to tackle any challenge in synthetic biology.
Throughout our project, we used standardized Golden Gate Assembly (GGA) Type 1-8 Overhangs, characterized by Lee et al., to provide system modularity and to easily build our DNA constructs (Figure 1) [3]. By designing each of our parts using GGA Type Overhangs, we broaden our flexibility when creating genetic circuits to integrate into ADP1. This modular system allows us to swap in any part with the same GGA Type Overhang to further explore ADP1's capabilities. By implementing this interchangeable system into our project, we contributed to the establishment of a more effective and standardized overhang system in the realm of synthetic biology. Through our experiments, we have demonstrated the robustness of using GGA Type Overhangs when engineering ADP1.
Fig. 1. Type 1-8 GGA Standardized Overhang Sequences. Adapted from [3].
Using this GGA Type 1-8 Overhang system, we constructed an elegant detector sequence (BBa_part) with the ability to swap parts in and out. Figure 2 illustrates the convertible nature of our system, highlighting the important GGA Type Overhangs and restriction sites that allow for GGA ligation. DNA parts highlighted in purple represent the upstream and downstream homology regions of our Pseudogymnoascus destructans target DNA sequence. These parts can be altered to detect any DNA sequence of interest as long as they contain the correct 4 bp GGA Type 1 and Type 7 Overhangs, described in Figure 1. See Figure 4 on the Engineering Page for more details on how to design primers containing the correct GGA Type Overhang and restriction sites.
Fig. 2. Detector Sequence Versatility with Flanking Homologies.
Using ADP1's natural competence and homologous recombination mechanism, our system can detect any DNA sequence with the matching upstream and downstream homology flanks. For more details on the detector accuracy and factors influencing transformation frequency, see the Model Page and Results Page. Due to the flexibility of our detector sequence and GGA Type Overhangs, we are not limited to only interchanging one part of the system. Figure 3 demonstrates how we can easily engineer four possible repressors that can be swapped into ADP1 to optimize our biosensor. Although we have not experimentally tested the four different repressors, testing each repressor and respective Yellow Fluorescent Protein (YFP) is important for characterizing expression in ADP1. Future iGEM teams can utilize our simple and intuitive modular system to test a variety of functional parts in ADP1.
Fig 3. Detector Sequence Versatility with Repressor Parts.
Figures 2 and 3 both depict alterations in our detector system, but further changes can be made given that a part contains the appropiate overhangs to ligate to a compatible sequential overhang. Additionally, our detector sequence leaves room for three additional parts to be inserted using Type 3, 4, and 5 overhangs, demonstrating the flexible and modular nature of the detector system. We have documented a strong foundational system for engineering ADP1, and we hope future iGEM teams will use our system to broaden our knowledge of ADP1 and its functionality as a synthetic biology chassis organism.
The most important contributional goals of the 2022 UT iGEM team go beyond our project goal of obtaining a viable biosensor to detect White Nose Syndrome in the environment using ADP1. While our research has the potential to have a direct positive impact on the environment, as corroborated by the public and professionals alike (See Human Practices Page)
The most impactful accomplishment that the 2022 UT Austin iGEM team has achieved this year involves our contribution to creating foundational understandings and intuitive implementation of ADP1 as an advantageous chassis organism in synthetic biology. The flexibility of our part designs, improvements to ADP1’s genome, and efficient modular protocols for transforming and engineering ADP1 have allowed us to play a significant role in advancing our understanding and utilization of a nascent organism in synthetic biology.
[1] Gomez, M. J., & Neyfakh, A. A. (2006). Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrobial agents and chemotherapy, 50(11), 3562-3567. https://doi.org/10.1128/AAC.00579-06
[2] Overballe-Petersen, S., Harms, K., Orlando, L. A., Mayar, J. V. M., Rasmussen, S., Dahl, T. W., ... & Willerslev, E. (2013). Bacterial natural transformation by highly fragmented and damaged DNA. Proceedings of the National Academy of Sciences, 110(49), 19860-19865. https://doi.org/10.1073/pnas.1315278110
[3] Lee, M.E., DeLoache, W.C., Cervantes, B., and Dueber, J.E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology 4, 975–986. 10.1021/sb500366v.
[4] Metzgar, D., Bacher, J. M., Pezo, V., Reader, J., Doring, V., Schimmel, P., Marliere, P., & de Crecy-Lagard, V. (2004). Acinetobacter sp.. ADP1: An ideal model organism for genetic analysis and Genome Engineering. Nucleic Acids Research, 32(19), 5780–5790. https://doi.org/10.1093/nar/gkh881.
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