ACIAD2049 Upstream

Introduction

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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 [1]. 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. Restriction sites are attached to the 3’ end, which are specifically designed to allow for ligation to the tdk/kan selection cassette (BBa_K4342000). This composite part, BBa_K4342019, then allows for the deletion of the ACIAD2049 gene via our ADP1 Golden Transformation protocol, found on this page.

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 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 part is designed to ligate to the 5' end of the tdk/kan cassette, BBa_K4342000, 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.

The tdk/kan cassette can then be deleted from this composite part to produce a scarless deletion of the ACIAD2049 gene (BBa_4342020), in accordance to our ADP1 Golden Transformation protocol.

BsmBI Restriction Site

The BsmBI site of this part is designed to ligate to the 5’ end of the ACIAD2049 Downstream Homology, (BBa_K4342010) creating the ΔACIAD2049 Homologies composite part (BBa_4342020). This composite part serves as a “rescue” cassette to select for successful transformants on Azidothymidine (AZT).

Characterization

References

[1] 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.