Part:BBa_K4342022
pbpG Rescue 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 Rescue is categorized as a Rescue cassette in our part collection. This part is not categorized by a GGA Type Overhang because this part is ligated via BsmBI digestion, rather than BsaI digestion.
Usage and Biology
pbpG is a gene in Acinetobacter baylyi ADP1 that encodes a penicillin-binding protein involved in peptidoglycan synthesis[1] It also contributes β-lactam antibiotic resistance [1]. Knocking out this gene allows for the integration of other DNA sequences in its chromosomal location. Using this part, we demonstrate that pbpG can be replaced with any DNA construct. Specifically, the pbpG Rescue cassette knocks out pbpG from ADP1.
Design
The pbpG Rescue part comprises the 2016 bp region combining the pbpG Upstream (BBa_4342011) 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 (BBa_4342021) 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 pbpG via BsmBI digestion. During this second-step transformation, we transform this pbpG Rescue cassette into ADP1 containing the pbpG Integration cassette (BBa_4342021). Using this part allows us to select for successful transformants on Azidothymidine (AZT) (Figure 2). Successful transformants have removed the tdk/kan cassette and integrated our pbpG Rescue cassette, resulting in the deletion of pbpG from ADP1.
Characterization
To confirm that we successfully created this part, we transformed the pbpG Rescue cassette into ADP1-ISx [3] containing the pbpG Integration cassette. We selected for successful transformants on LB-AZT plates and picked colonies to inoculate and store frozen stocks of our newly constructed deletion strain.
References
[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] 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] 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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1339
Illegal PstI site found at 1283 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1283
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1339
Illegal PstI site found at 1283 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1339
Illegal PstI site found at 1283 - 1000COMPATIBLE WITH RFC[1000]
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