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 (BBa_4342003) and (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 (BBa_4342002) and (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. Examples include (BBa_4342019) and (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. Examples include (BBa_4342020, Upstream + Downstream), (BBa_4342030, Upstream + Genetic Device + Downstream), and (BBa_4342031, Upstream + Composite + Downstream).

ACIAD2049 Upstream Type 1 is characterized as an Upstream basic part in our part collection.

We further characterize each part with a 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 are characterized with multiple numbers, such as Type 234. These DNA sequences start with a Type 2 Overhang and ends with a Type 4 Overhang (ex. tdk/kan cassette (BBa_4342000).

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 a mutated nptII gene 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 Type 1 part comprises the 1292 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. See Figure 4 on the Engineering Page for more details on how to design primers containing the correct GGA Type Overhang and restriction sites.

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 GGA Type Overhangs. 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.

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 tdk/kan cassette composite part (BBa_4342019). This composite part allows for successful transformant selection on Kanamycin (Kan) via the kanR gene (Fig. 1).

Fig. 1. The insertion of the tdk/kan cassette in place of a target gene (ACIAD2049).

Step 2

The tdk/kan cassette can subsequently be knocked out to create a scarless deletion of ACIAD2049 via BsmBI digestion, BBa_4342020. During this reaction, this part is ligated to the 5' end of the ACIAD2049 Downstream part BBa_4342002. This composite part serves as a “rescue” cassette to select for successful transformants on Azidothymidine (AZT) (Fig. 2).

Fig. 2. The scarless deletion of the tdk/kan cassette produced by BsmBI digestion.

Experimental Data

To confirm that we successfully created this part, we performed a PCR and gel electrophoresis using genomic DNA from the ADP1-ISx strain 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 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.