Part:BBa_K4342001
ACIAD2049 Upstream
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 - A "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 - A "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).
ACIAD2049 Upstream is categorized as a Type 1 Upstream basic part in our part collection.
Usage and Biology
ACIAD2049 is a nonessential gene in Acinetobacter baylyi ADP1 [1]. Knocking out this gene allows for the integration of other DNA sequences in its chromosomal location. Using this part, we demonstrate that ACIAD2049 can be replaced with any DNA construct. Specifically, we have inserted an nptII Broken Gene (BBa_4342015) in place of ACIAD2049 to detect the presence of a Wild-Type nptII gene, showing how ADP1 can be engineered to detect antibiotic resistance.
Design
The ACIAD2049 Upstream part comprises the 1277 bp homology directly upstream of the ACIAD2049 gene in ADP1. We designed optimized primers, which include GC contents of over 40% and melting temperatures of under 70°C. BsaI and BsmBI restriction sites are attached to the 3’ end, which are designed to ligate to the 5' end of the tdk/kan cassette (BBa_4342000) and the ACIAD2049 Downstream part (BBa_4342002) respectively.
This part contains a BsaI restriction site with a standard 4 bp GGA Type 2 Prefix [2] and a BsmBI restriction site with a 4 bp “rescue” complementary scar. See the Contribution page on our wiki for more details on how GGA Type Overhangs provide system modularity. This design allows for easy ligation with any part that contains a complementary 4 bp GGA Type 2 Prefix (BsaI) or the same 4 bp “rescue” complementary scar.
- 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.
Composite Parts
This basic part is used to assemble the ACIAD2049 Integration cassette (BBa_4342019) and the ACIAD2049 Rescue cassette (BBa_4342020) composite parts. Figures 1 and 2 show how these composite parts can be used in our two-step ADP1 Genetic Engineering protocol to create a minimal 4 bp scar in the deletion of the ACIAD2049 gene.
Step 1
This part is designed to ligate to the 5' end of the tdk/kan cassette, BBa_4342000, creating the ACIAD2049 Integration cassette composite part (BBa_4342019). This composite part allows 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, this part is ligated to the 5' end of the ACIAD2049 Downstream (BBa_4342002). part. This reaction assembles the ACIAD2049 Rescue cassette (BBa_4342020) to select for successful transformants on Azidothymidine (AZT) (Figure 2).
Characterization
To confirm that we successfully created this part, we performed a PCR and gel electrophoresis using genomic DNA from the ADP1-ISx strain [3] as a template. Bands were visible at ~1300 bp, confirming the amplification of the ACIAD2049 Upstream part. A PCR master mix with diH2O in place of template DNA was used as a negative control.
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] 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
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1040
Illegal XbaI site found at 180
Illegal PstI site found at 645 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1040
Illegal PstI site found at 645 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1040
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1040
Illegal XbaI site found at 180
Illegal PstI site found at 645 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1040
Illegal XbaI site found at 180
Illegal PstI site found at 645 - 1000COMPATIBLE WITH RFC[1000]
| None |