Part:BBa_K4342032

TEM-1 Detector 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).

Usage and Biology

acrB is a gene in Acinetobacter baylyi ADP1 which codes for proteins involved with efflux pumps [1]. acrB also contributes to intrinsic 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 acrB can be replaced with any DNA construct. Specifically, we have inserted a mutated TEM-1 gene (BBa_4342017) in place of acrB to detect the presence of a Wild-Type TEM-1 gene, showing how ADP1 can be engineered to detect antibiotic resistance

The TEM-1 gene in Acinetobacter baylyi ADP1 codes for β-lactamase resistance. Deleting a section of this gene prevents ADP1 from growing on β-lactams, making ADP1 susceptible to this family of antibiotics. This allows for the detection of homologous recombination/transformation of the correct TEM-1 β-lactamase resistance gene by ADP1 [2],[3].

Design

The TEM-1 detector rescue cassette is comprised of the 3128 base pairs. The composite part combines the acrB Upstream (BBa_4342009) + the TEM-1 broken gene (BBa_4342017) + the acrB Downstream (BBa_4342010) creating the TEM-1 detector rescue cassette (BBa_4342032). This composite part permits the selection of transformants through β-lactamase resistance.

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] De Vries, J., Wackernagel, W. (1998). Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation. Molecular and General Genetics, 257, 606-613. https://doi.org/10.1007/s004380050688

[3] Kong, K., Schneper, L., Mathee, K. (2010). Beta-lactam Antibiotics: From Antibiosis to Resistance and Bacteriology. APMIS, 118(1), 1-36. https://doi.org/10.1111/j.1600-0463.2009.02463.x