Part:BBa_K4342021
pbpG Integration Cassette
Introduction
The 2022 UT Austin iGEM Team’s Part Collection 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 this 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 hope this part collection guides future iGEM teams in engineering ADP1 and utilizing ADP1’s flexibility to tackle any challenge in synthetic biology.
Categorization
For our parts collection, we categorize our parts into the following categories:
Upstream
An Upstream basic part is a DNA sequence directly upstream of a target gene. These basic parts are homology flanks that are used for ADP1 Genetic Engineering. Examples include the ACIAD2049 Upstream for P. destructans detector (BBa_4342003) and pbpG Upstream (BBa_4342011).
Downstream
A Downstream basic part is a DNA sequence directly downstream of a target gene. These basic parts are homology flanks that are used for ADP1 Genetic Engineering. Examples include ACIAD2049 Downstream for P. destructans detector (BBa_4342004) and pbpG Downstream (BBa_4342012).
Integration Cassettes
An "Integration" cassette is a composite part consisting of an "Upstream" basic part, the tdk/kan basic part (BBa_4342000), and a "Downstream" basic part. These parts are designed to use in the first transformation step in ADP1 Genetic Engineering. Examples include the ACIAD2049 Integration cassette (BBa_4342019) and the acrB Integration cassette (BBa_4342023).
Rescue Cassettes
"Rescue" cassette is a composite part consisting of an "Upstream" basic part, an optional genetic device, and a "Downstream" basic part. These parts are designed to use in the second transformation step in ADP1 Genetic Engineering. Examples include the ACIAD2049 Rescue cassette (BBa_4342020, Upstream + Downstream), the YFP Rescue cassette (BBa_4342030, Upstream + Genetic Device + Downstream), and the nptII Detector Rescue cassette (BBa_4342031, Upstream + Composite Part + Downstream).
Genetic Device
"Genetic Device" is a basic part that can be any DNA sequence to be integrated into ADP1. Examples include the CymR YFP (BBa_4342008) and the nptII Broken Gene (BBa_4342015).
We further categorize each part with a standardized Golden Gate Assembly (GGA) Type 1-8 Overhang [2]. Each type is ligated to a complementary type (ex. Type 2 can be ligated to Type 1 and Type 3). Moreover, some parts contain consecutive GGA Type numbers, such as Type 234. These DNA sequences start with a Type 2 Overhang and end with a Type 4 Overhang (ex. tdk/kan cassette (BBa_4342000).
pbpG Integration is categorized as a Type 1-5 Integration cassette in our part collection.
Usage and Biology
pbpG is a gene in Acinetobacter baylyi ADP1 that contributes to intrinsic β-lactam antibiotic resistance [2]. pbpG encodes a penicillin-binding protein involved in peptidoglycan synthesis. Knocking out this gene makes ADP1 more susceptible to β-lactam antibiotics, such as Ampicillin. pbpG also provides an additional ADP1 chromosomal location where other DNA sequences can be integrated. Using this part, we demonstrate that pbpG can be deleted from ADP1's genome. Further data on the Results Page show that we successfully made ADP1 more susceptible to β-lactam antibiotics.
Design
The pbpG Integration part comprises the 3695 bp region combining the pbpG Upstream (BBa_4342011), tdk/kan (BBa_4342000) cassette, and pbpG Downstream (BBa_4342012) parts.
- Please note that BsaI restriction sites have been removed to meet RFC[1000] BioBrick Assembly Compatibility. To see in-depth primer design, please see Figure 4 on the Engineering Page for more details on how to design primers containing the correct GGA Type Overhang and restriction sites.
Step 1
The pbpG Integration cassette is designed to allow for successful transformant selection on Kanamycin (Kan) via the kanR gene (Figure 1).
Step 2
The tdk/kan cassette can subsequently be knocked out to create a 4 bp minimal scar deletion of ACIAD2049 via BsmBI digestion. During this reaction, a pbpG Rescue cassette (BBa_4342022) is constructed. We use the pbpG Rescue cassette to select for successful transformants on Azidothymidine (AZT) (Figure 2).
Characterization
To confirm that we successfully created this part, we transformed the pbpG Integration cassette into ADP1-ISx [3]. First, we ran gel electrophoresis on our unpurified GGA product, containing the pbpG Integration cassette (Figure 3). Lane 1 contains our pbpG Integration cassette, shown by the 3.7 kb tdk/kan ligated to homology flanks. There are some background bands present in the lanes, including a 3.3 kb pBTK622 plasmid, 1.7 kb tdk/kan cassette, and 1 kb homology flanks.
After we confirmed the part was present in the GGA reaction, we proceeded with first-step integration of the tdk/kan cassette and selected for successful transformants on LB-Kan plates (Figure 4). This figure demonstrates that the DNA sequence was integrated, and the pbpG gene was replaced with the tdk/kan cassette. Thus, the first-step integration was successfully completed.
For step 2, we picked colonies to inoculate for second-step removal of the tdk/kan cassette. Second-step removal requires the use of a rescue cassette.
References
[1] Suárez, G.A., Dugan, K.R., Renda, B.A., Leonard, S.P., Gangavarapu, L.S., and Barrick, J.E. (2020). Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining. Nucleic Acids Research 48, 4585–4600. 10.1093/nar/gkaa204.
[2] 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.
[3] 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
[4] Suárez, G. A., Renda, B. A., Dasgupta, A., & Barrick, J. E. (2017). Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx. Applied and environmental microbiology, 83(17), e01025-17. https://doi.org/10.1128/AEM.01025-17
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