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.

The most important DNA sequence for ADP1 engineering is the tdk/kan cassette [1] (BBa_K4342000), shown in Figure 1. We use the tdk/kan cassette to select for successful ADP1 transformants. The kanR gene allows us to select on Kanamycin (Kan), and the tdk gene allows us to select on Azidothymidine (AZT). We describe a reliable two-step ADP1 Genetic Engineering protocol using this fundamental tdk/kan cassette. The first ADP1 transformation inserts the tdk/kan cassette into ADP1's chromosome, deleting the target sequence. The second step removes the tdk/kan cassette from the genome by "rescuing" ADP1 from AZT susceptibility. This second step is when we can insert a genetic device capable of detecting environmental DNA (eDNA).

Fig. 1. tdk/kan Cassette. tdk confers Azidothymidine (AZT) susceptibility, and kanR confers Kanamycin (Kan) susceptibility.

This part collection contains 5 different part types, all of which are compatible with the ADP1 transformation protocol. These part types include:

• • Upstream Part - A DNA sequence that is identical to a region of the ADP1 genome directly upstream of a gene.
• • Downstream Part - A DNA sequence that is identical to a region of the ADP1 genome directly downstream of a gene.
• • Integration Cassette - A DNA sequence that contains the tdk/kan cassette.
• • Rescue Cassette - A DNA sequence designed to remove the tdk/kan cassette.
• • Genetic Device - Any DNA sequence that can be inserted into ADP1's genome such as a GFP gene.

ADP1 Genetic Engineering

Step 1. Integration of tdk/kan Selection Casette

The first step to engineering ADP1 is inserting the tdk/kan cassette into ADP1's chromosome via Kan selection. The parts required for this step include an Upstream part, a Downstream part, and the tdk/kan cassette. These parts can be combined to make an integration cassette, which is a composite part designed to integrate the tdk/kan cassette into the ADP1's chromosome. Figure 2 shows an integration cassette transforming ADP1, knocking out the target gene and replacing the target gene with the tdk/kan cassette. Cells that successfully integrated the tdk/kan cassette can be selected for by plating on LB-Kan. The following composite parts are integration cassettes made from different combinations of Upstream and Downstream parts.

• • ACIAD2049 Integration Cassette (BBa_K4342019)
• • pbpG Integration Cassette (BBa_K4342021)
• • acrB Integration Cassette (BBa_K4342023)
• • recJ Integration Cassette (BBa_K4342025)

Fig. 2. Integration of tdk/kan Selection Cassette.

Step 2. Removal of the tdk/kan Selection Casette

a. Knocking out an ADP1 Gene

The second step to engineering ADP1 is removing the tdk/kan cassette via AZT counterselection. The parts required for this step include an Upstream part and a Downstream part. The Upstream and Downstream parts can be combined to create a rescue cassette , which is a composite part used to knockout the tdk/kan cassette. Figure 3 shows a rescue cassette transformed into an ADP1 strain that contains the tdk/kan cassette, removing the tdk/kan cassette and producing a 4 bp minimal scar deletion of the target gene. Because the tdk gene is lethal to ADP1 in the presence of AZT, plating on LB-AZT selects for ADP1 cells that knocked out the tdk/kan cassette. The following composite parts are some of the rescue cassettes we used during our project.

• • ACIAD2049 Rescue Cassette (BBa_K4342020)
• • pbpG Rescue Cassette (BBa_K4342022)
• • acrB Rescue Cassette (BBa_K4342024)
• • recJ Rescue Cassette (BBa_K4342026)

Fig. 3. Removal of the tdk/kan Selection Cassette.

b. Inserting a Genetic Device

Step 2 of this protocol can also be used to insert any genetic device into ADP1's genome by removing the tdk/kan cassette. This process requires creating a rescue cassette that contains a genetic device in between the Upstream and Downstream parts. Figure 4 shows how Step Two of this protocol uses a rescue cassette to replace the tdk/kan cassette with a genetic device. Similarly to the Step 2a transformation, plating on LB-AZT selects for cells that possess the genetic device. We used this step of the engineering protocol to insert the nptII and TEM-1 detector construct parts into ADP1's genome. The following composite parts are some of the rescue cassettes that contain genetic devices we used during our project.

• • nptII Rescue Cassette (BBa_K4342031)
• • TEM-1 Rescue Cassette (BBa_K4342032)

Fig. 4. Inserting a Genetic Device via tdk/kan Counter-Selection.

Best New Basic Part

The most important DNA sequence for ADP1 engineering is the tdk/kan cassette [1] (BBa_K4342000), our nomination for this year’s best new part. The tdk/kan cassette uses the kanR gene for selection and the tdk gene for counterselection in order to select for successful ADP1 transformants. In conjunction with the two-step engineering protocol described above, this part can be used by future iGEM teams to efficiently modify ADP1's genome. We used this part to successfully knock out multiple nonessential ADP1 genes, insert several genetic devices, and build multiple biosensors for the detection of exogenous DNA. We hope that future iGEM teams draw inspiration from our work and use the tdk/kan cassette to engineer ADP1 to solve real-world problems.

Best New Composite Part

We nominate our acrB Integration Cassette (BBa_K4342023) for the Best New Composite Part award because we believe future iGEM teams can use it to engineer ADP1 in novel ways. This part consists of the acrB Upstream part (BBa_K4342009), the tdk/kan cassette (BBa_K4342000), and an acrB Downstream part (BBa_K4342010) ligated together. The acrB Integration Cassette is integral to our part collection because this composite part provides the sequence necessary to delete or replace the acrB gene. We identified the acrB gene location as being an ideal location for genome engineering because acrB is nonessential and contributes to ADP1's intrinsic β-lactam antibiotic resistance [2]. Using this part, we have successfully created an ADP1-based biosensors, and we hope that future iGEM teams will be able to expand upon this part's potential.

Best Part Collection

We nominate our part collection (BBa_K4342000 - BBa_K4342033) for this year’s Best Part Collection Award, because it contains all the tools one could need to engineer Acinetobacter baylyi ADP1. This collection includes: the tdk/kan cassette, which is essential for confirming the engineering of ADP1 via selection and counterselection, Upstream and Downstream parts which flank the gene that will be replaced and/or deleted, and Integration and Rescue cassettes which assist in deleting ADP1 genes and inserting genetic devices. In conjunction with our two-step ADP1 Genetic Engineering protocol, we hope this part collection will be used by future iGEM teams to take advantage of ADP1 as a chassis organism for synthetic biology.

References