Part:BBa_K4342001

ACIAD2049 Upstream

Usage and Biology

ACIAD2049 is a nonessential gene in Acinetobacter baylyi ADP1. Knocking out this gene allows for the integration of other DNA sequences in its chromosomal location. Cooper et al. and colleagues 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 deleting ACIAD2049 can be used to detect antibiotic resistance genes using ADP1 as a chassis organism.

Design

The ACIAD2049 Upstream Homology part comprises the 1292 bp region directly upstream of the ACIAD2049 gene in ADP1. Restriction sites are attached to the 3’ end, which are specifically designed to allow for the deletion of the ACIAD2049 gene through our two-step ADP1 Golden Transformation protocol using the tdk/kan selection cassette. This protocol utilizes PCR and Golden Gate Assembly (GGA) to construct sequences necessary for deleting ACIAD2049.

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.

???? Additionally, this part is classified as a Type 1a part in our parts collection (BBa_K4342000-BBa_K4334033), since it is an upstream homology required to create the tdk/kan integration cassette. This integration cassette allows for the selection of engineered ADP1 with kanamycin. ????

BsaI Restriction Site

The BsaI site of 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.

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).

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.