Difference between revisions of "Part:BBa K4342001"
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<h1>Design</h1> | <h1>Design</h1> | ||
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| − | + | 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 is designed to ligate to any sequence possessing the complementary overhang. The ACIAD2049 Upstream part is included in the ACIAD2049 integration cassette (BBa_) and ACIAD2049 rescue cassette (BBa_) composite parts which are used for deleting the ACIAD2049 gene via our ADP1 Golden Transformation protocol, found on [https://2022.igem.wiki/austin-utexas/parts this page]. | |
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| − | 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 | + | |
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 [https://2022.igem.wiki/austin-utexas/contribution 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. | 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 [https://2022.igem.wiki/austin-utexas/contribution 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. | ||
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===Step 1=== | ===Step 1=== | ||
This part is designed to ligate to the 5' end of the <em> tdk/kan </em> cassette, [https://parts.igem.org/Part:BBa_K4342000 BBa_4342000], creating the ACIAD2049 <em> tdk/kan </em> cassette composite part [https://parts.igem.org/Part:BBa_K4342019 (BBa_4342019)]. This composite part allows for successful transformant selection on Kanamycin (Kan) via the <i>kanR</i> gene (Fig. 1). | This part is designed to ligate to the 5' end of the <em> tdk/kan </em> cassette, [https://parts.igem.org/Part:BBa_K4342000 BBa_4342000], creating the ACIAD2049 <em> tdk/kan </em> cassette composite part [https://parts.igem.org/Part:BBa_K4342019 (BBa_4342019)]. This composite part allows for successful transformant selection on Kanamycin (Kan) via the <i>kanR</i> gene (Fig. 1). | ||
| − | + | [[File:TdkKan_Selection.png|500px|thumb|center|The insertion of the <i>tdk/kan</i> cassette in place of a target gene (ACIAD2049).]] | |
===Step 2=== | ===Step 2=== | ||
| − | The <i>tdk/kan</i> cassette can subsequently be knocked out to create a scarless deletion of ACIAD2049 via BsmBI digestion, [https://parts.igem.org/Part:BBa_K4342020 BBa_4342020]. During this reaction, this part is ligated to the 5' end of the ACIAD2049 Downstream part [https://parts.igem.org/Part:BBa_K4342002 BBa_4342002]. This composite part serves as a “rescue” cassette to select for successful transformants on Azidothymidine (AZT) (Fig. 2). | + | The <i>tdk/kan</i> cassette can subsequently be knocked out to create a scarless deletion of ACIAD2049 via BsmBI digestion, [https://parts.igem.org/Part:BBa_K4342020 BBa_4342020]. During this reaction, this part is ligated to the 5' end of the ACIAD2049 Downstream part [https://parts.igem.org/Part:BBa_K4342002 BBa_4342002]. This composite part serves as a “rescue” cassette to select for successful transformants on Azidothymidine (AZT) (Fig. 2). [[File:TdkKan_Counterselection.png|500px|thumb|center|The scarless deletion of the <i>tdk/kan</i> cassette produced by BsmBI digestion.]] |
<h1>Characterization</h1> | <h1>Characterization</h1> | ||
Revision as of 17:53, 11 October 2022
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 [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 is designed to ligate to any sequence possessing the complementary overhang. The ACIAD2049 Upstream part is included in the ACIAD2049 integration cassette (BBa_) and ACIAD2049 rescue cassette (BBa_) composite parts which are used for deleting 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 basic part can be assembled to create composite parts using the BsaI restriction site, which can be then used to integrate the tdk/kdan cassette in place of the ACIAD2049 gene (Fig. 1). Then, BsmBI digestion can be used with this part to create scarless deletions of the ACIAD2049 gene (Fig. 2).
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).
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).
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] 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.