Part:BBa_K4342001

ACIAD2049 Upstream

Introduction

The 2022 UT Austin iGEM Team’s Part Collection provides clear and reliable protocols for genetically engineering Acinetobacter baylyi ADP1. On our Parts page, we explain how our part collection can be used alongside a two-step Golden Transformation 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.

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. Cooper et al. have taken advantage of the ACIAD2049 gene deletion to create ADP1-based biosensors capable of detecting diseases within the human body [2]. Using this part, we demonstrate that ACIAD2049 knockouts can be used to detect antibiotic resistance genes using ADP1 as a chassis organism.

Design

The ACIAD2049 Upstream part comprises the 1292 bp homology directly upstream of the ACIAD2049 gene in ADP1. This specific region was chosen to create 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 Downsteam part (BBa_4342002) respectively.

This part contains a BsaI restriction site with a standard 4 bp GGA Type 2 Prefix 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 engineering protocol to create a scarless 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.

Characterization

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] Cooper, R. M., Wright, J. A., Ng, J. Q., Goyne, J. M., Suzuki, N., Lee, Y. K., Ichinose, M., Radford, G., Thomas, E. M., Vrbanac, L., Knight, R., Woods, S. L., Worthley, D. L., & Hasty, J. (2021). Engineered bacteria detect tumor DNA in vivo. bioRxiv. https://doi.org/10.1101/2021.09.10.459858.