cymR YFP

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).

cymR YFP is categorized as a Type 2-4 Genetic Device basic part in our part collection.

Usage and Biology

The CymR YFP in Acinetobacter baylyi ADP1 codes for a yellow fluorescent protein.

Design

The CymR YFP part comprises the 1014 base pair region in place of the acrB gene of ADP1. This part has BsaI restriction sites attached to the 5’ end and 3’ ends, which are specifically designed to ligate to the acrB Upstream Homology (BBa_4342009) and the acrB Downstream Homology (BBa_4342010)via BsaI digestion in the P. destructans Detector strain.

BsaI Restriction Sites

The BsaI sites are designed to ligate to the 3’ end of the acrB Upstream Homology (BBa_4342009) and the 5’ end of the acrB Downstream Homology (BBa_4342010) This basic part permits the expression of a YFP gene using this promoter, which allows for screenable selection when the CymR repressor (BBa_4342007) is deleted through homologous recombination of P. destructans eDNA.

Characterization

Fig. 3. YFP Insertions. From left to right: Ladder, Control, ADP1-ISx, cymR-YFP, vanR-YFP, lacI-YFP, and betI-YFP. Expected length for each YFP variation was ~3000 base pairs.

References

[1] Meyer, A., Shegall-Shapiro, T., Glassey, E., Zhang, J., Voigt, C. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small molecule sensors. Nature Chemical Biology 15, 196-204 (2019). https://doi.org/10.1038/s41589-018-0168-3

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]