Difference between revisions of "Part:BBa K4342000"
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<partinfo>BBa_K4342000 short</partinfo>
 
<partinfo>BBa_K4342000 short</partinfo>
  
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<h1>Usage and Biology</h1>
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===Introduction===
 +
The 2022 UT Austin iGEM Team’s Part Collection provides a number of DNA sequences and procedures for genetically engineering <i>Acinetobacter baylyi </i> ADP1. We were able to effectively engineer ADP1's genome using a two-step genetic engineering protocol. See the [https://2022.igem.wiki/austin-utexas/engineering 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. <b>We hope this part collection guides future iGEM teams in engineering ADP1 and utilizing ADP1’s flexibility to tackle any challenge in synthetic biology.</b>
  
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The <i>tdk/kan</i> cassette is a highly versatile sequence used for efficiently and effectively engineering the <i> Acinetobacter baylyi </i> ADP1 genome. This part contains the <i> kanR </i> and <i> tdk </i> genes which allow for the selection and counterselection of genetically engineered ADP1 cells. When this cassette is integrated into the ADP1 genome, the <i> kanR </i> gene confers resistance to kanamycin and the <i> tdk </i> gene is lethal to the cell when Azidothymidine is present (Metzgar et. al - 2004).
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===Categorization===
 +
For our parts collection, we categorize our parts into the following categories:
  
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The 2022 UT Austin iGEM team used the <i> tdk/kan </i> cassette to successfully delete a number of genes, insert DNA sequences into the ADP1 chromosome, and engineer an ADP1-based biosensor for detecting any DNA sequence of interest. The composite parts below utilized the <i> tdk/kan </i> cassette for genetic engineering.  
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<b> Upstream </b> - An <b> Upstream </b> 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 the ACIAD2049 Upstream for <i>P. destructans</i> detector [https://parts.igem.org/Part:BBa_K4342003 (BBa_4342003)] and <i>pbpG</i> Upstream [https://parts.igem.org/Part:BBa_K4342011 (BBa_4342011)].
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<ul>
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<li> <i> ACIAD2049 tdk/kan </i> Cassette </li>
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<li> <i> ACIAD2049 </i> Rescue Cassette</li>
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<li> <i> AcrB tdk/kan </i> Cassette </li>
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<li> <i> AcrB </i> Rescue Cassette </li>
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<li> <i> PbpG tdk/kan Cassette </i> </li>
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<li> <i> PbpG </i> Rescue Cassette </li>
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<li> <i> RecJ tdk/kan </i> Cassette </li>
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<li> <i> RecJ </i> Rescue Cassette </li>
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<li> Broken <i> nptII </i> Gene </li>
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<li> <i> nptII </i> Gene Detector Construct</li>
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<li> <i> TEM-1 </i> Broken Gene </li>
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<li> <i> TEM-1 </i> Gene Detector Construct</li>
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 +
<b> Downstream </b> - A <b> Downstream </b> 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 ACIAD2049 Downstream for <i>P. destructans</i> detector [https://parts.igem.org/Part:BBa_K4342004 (BBa_4342004)] and <i>pbpG</i> Downstream [https://parts.igem.org/Part:BBa_K4342012 (BBa_4342012)].
  
 +
<b> Integration Cassettes </b> - An <b> "Integration" cassette </b> is a composite part consisting of an "Upstream" basic part, the <i>tdk/kan</i> basic part [https://parts.igem.org/Part:BBa_K4342000 (BBa_4342000)], and a "Downstream" basic part. These parts are designed to use in the first transformation step in ADP1 Genetic Engineering. Examples include the ACIAD2049 Integration cassette [https://parts.igem.org/Part:BBa_K4342019 (BBa_4342019)] and the <i>acrB</i> Integration cassette [https://parts.igem.org/Part:BBa_K4342023 (BBa_4342023)].
  
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<b> Rescue Cassettes </b> - A <b> "Rescue" cassette </b> is a composite part consisting of an "Upstream" basic part, an optional genetic device, and a "Downstream" basic part. These parts are designed to use in the second transformation step in ADP1 Genetic Engineering. Examples include the ACIAD2049 Rescue cassette [https://parts.igem.org/Part:BBa_K4342020 (BBa_4342020], Upstream + Downstream), the YFP Rescue cassette [https://parts.igem.org/Part:BBa_K4342030 (BBa_4342030], Upstream + Genetic Device + Downstream), and the <i>nptII</i> Detector Rescue cassette [https://parts.igem.org/Part:BBa_K4342031 (BBa_4342031], Upstream + Composite Part + Downstream).
  
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<h1>Design</h1>
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<b> Genetic Device </b> - A <b>"Genetic Device"</b> is a basic part that can be any DNA sequence to be integrated into ADP1. Examples include the <i>CymR</i> YFP [https://parts.igem.org/Part:BBa_K4342008 (BBa_4342008)] and the <i>nptII</i> Broken Gene  [https://parts.igem.org/Part:BBa_K4342015 (BBa_4342015)].
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As seen in figure x, the tdk/kan cassette is flanked by bsaI restriction sites on both its 5’ and 3’ ends. These restriction sites produce sticky ends which can ligate to any DNA sequence possessing the necessary restriction sites with the complementary overhangs. Figure X shows how the tdk/kan cassette can be ligated to DNA sequences homologous to ADP1’s genome.
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<b> ACIAD2049 Upstream </b> is categorized as an <b> Upstream </b> basic part in our part collection.
 +
 
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We further categorize each part with a standardized Golden Gate Assembly (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 contain consecutive GGA Type numbers, such as Type 234. These DNA sequences start with a Type 2 Overhang and end with a Type 4 Overhang (ex. <i>tdk/kan</i> cassette [https://parts.igem.org/Part:BBa_K4342000 (BBa_4342000)].
 +
 
 +
===Usage and Biology===
 +
 
 +
The <i>tdk/kan</i> cassette is the keystone of our parts collection. It is a highly versatile sequence used for efficiently and effectively engineering the <i>Acinetobacter baylyi</i> ADP1 genome. This part contains the <i>kanR</i> and <i>tdk</i> genes, which allow for the selection and counterselection of genetically engineered ADP1 cells. When this cassette is integrated into the ADP1 genome, the <i>kanR</i> gene confers resistance to kanamycin and the <i>tdk</i> gene is lethal to the cell when Azidothymidine is present [1].
 +
 
 +
The 2022 UT Austin iGEM team used the <i>tdk/kan</i> cassette to successfully delete a number of genes, insert DNA sequences into the ADP1 chromosome, and engineer an ADP1-based biosensor for detecting antibiotic resistance and <i>P. destructans</i>. Our design process and two-step ADP1 genetic engineering protocol can be found on the [https://2022.igem.wiki/austin-utexas/parts Parts], [https://2022.igem.wiki/austin-utexas/contribution Contribution], and [https://2022.igem.wiki/austin-utexas/engineering Engineering] pages of our Wiki.
 +
 
 +
===Design===
 +
The <b><i>tdk/kan</i> cassette </b> is a Type 234 part [2] featuring BsaI restriction sites on its 3' and 5' ends, allowing it to ligate to Upstream and Downstream parts in from our collection. The composite parts below utilized this part for genetic engineering:
 +
 
 +
<li> [https://parts.igem.org/Part:BBa_K4342019 ACIAD2049 Integration Cassette (BBa_K4342019)] </li>
 +
<li>[https://parts.igem.org/Part:BBa_K4342020 ACIAD2049 Rescue Cassette] (BBa_K4342020)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342021 PbpG Integration Cassette] (BBa_K4342021)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342022 PbpG Rescue Cassette] (BBa_K4342022)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342023 AcrB Integration Cassette] (BBa_K4342023)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342024 AcrB Rescue Cassette] (BBa_K4342024)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342025 RecJ Integration Cassette] (BBa_K4342025)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342026 RecJ Rescue Cassette] (BBa_K4342026)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342031 nptII Gene Detector Rescue Cassette] (BBa_K4342031)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342032 TEM-1 Gene Detector Rescue Cassette] (BBa_K4342032)</li>
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<li>[https://parts.igem.org/Part:BBa_K4342029 <i>P. destructans </i> Integration Cassette (BBa_K4342029)]
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Figures 1 and 2 demonstrate how our <b><i>tdk/kan</i> cassette </b> can be used in our two-step ADP1 Genetic Engineering protocol.
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[[File:TdkKan_Selection.png|500px|thumb|center|<b> Fig. 1. Step 1.</b> The insertion of the <i>tdk/kan</i> cassette in place of a target gene (ACIAD2049).]]
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[[File:TdkKan_Counterselection.png|500px|thumb|center|<b> Fig. 2. Step 2. </b> The scarless deletion of the <i>tdk/kan</i> cassette produced by BsmBI digestion.]]
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 +
===Characterization===
 +
(DATA HERE!)
 +
 
 +
[1] Metzgar, D., Bacher, J. M., Pezo, V., Reader, J., Doring, V., Schimmel, P., Marliere, P., & de Crecy-Lagard, V. (2004). Acinetobacter sp.. ADP1: An ideal model organism for genetic analysis and Genome Engineering. Nucleic Acids Research, 32(19), 5780–5790. https://doi.org/10.1093/nar/gkh881.
 +
 
 +
[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.

Revision as of 06:31, 12 October 2022

tdk/kan Cassette

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 the ACIAD2049 Upstream for P. destructans detector (BBa_4342003) and pbpG Upstream (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 ACIAD2049 Downstream for P. destructans detector (BBa_4342004) and pbpG Downstream (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. These parts are designed to use in the first transformation step in ADP1 Genetic Engineering. Examples include the ACIAD2049 Integration cassette (BBa_4342019) and the acrB Integration cassette (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. These parts are designed to use in the second transformation step in ADP1 Genetic Engineering. Examples include the ACIAD2049 Rescue cassette (BBa_4342020, Upstream + Downstream), the YFP Rescue cassette (BBa_4342030, Upstream + Genetic Device + Downstream), and the nptII Detector Rescue cassette (BBa_4342031, Upstream + Composite Part + Downstream).

Genetic Device - A "Genetic Device" is a basic part that can be any DNA sequence to be integrated into ADP1. Examples include the CymR YFP (BBa_4342008) and the nptII Broken Gene (BBa_4342015).

ACIAD2049 Upstream is categorized as an Upstream basic part in our part collection.

We further categorize each part with a standardized Golden Gate Assembly (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 contain consecutive GGA Type numbers, such as Type 234. These DNA sequences start with a Type 2 Overhang and end with a Type 4 Overhang (ex. tdk/kan cassette (BBa_4342000).

Usage and Biology

The tdk/kan cassette is the keystone of our parts collection. It is a highly versatile sequence used for efficiently and effectively engineering the Acinetobacter baylyi ADP1 genome. This part contains the kanR and tdk genes, which allow for the selection and counterselection of genetically engineered ADP1 cells. When this cassette is integrated into the ADP1 genome, the kanR gene confers resistance to kanamycin and the tdk gene is lethal to the cell when Azidothymidine is present [1].

The 2022 UT Austin iGEM team used the tdk/kan cassette to successfully delete a number of genes, insert DNA sequences into the ADP1 chromosome, and engineer an ADP1-based biosensor for detecting antibiotic resistance and P. destructans. Our design process and two-step ADP1 genetic engineering protocol can be found on the Parts, Contribution, and Engineering pages of our Wiki.

Design

The tdk/kan cassette is a Type 234 part [2] featuring BsaI restriction sites on its 3' and 5' ends, allowing it to ligate to Upstream and Downstream parts in from our collection. The composite parts below utilized this part for genetic engineering:

  • ACIAD2049 Integration Cassette (BBa_K4342019)
  • ACIAD2049 Rescue Cassette (BBa_K4342020)
  • PbpG Integration Cassette (BBa_K4342021)
  • PbpG Rescue Cassette (BBa_K4342022)
  • AcrB Integration Cassette (BBa_K4342023)
  • AcrB Rescue Cassette (BBa_K4342024)
  • RecJ Integration Cassette (BBa_K4342025)
  • RecJ Rescue Cassette (BBa_K4342026)
  • nptII Gene Detector Rescue Cassette (BBa_K4342031)
  • TEM-1 Gene Detector Rescue Cassette (BBa_K4342032)
  • P. destructans Integration Cassette (BBa_K4342029) Figures 1 and 2 demonstrate how our tdk/kan cassette can be used in our two-step ADP1 Genetic Engineering protocol.
    Fig. 1. Step 1. The insertion of the tdk/kan cassette in place of a target gene (ACIAD2049).
    Fig. 2. Step 2. The scarless deletion of the tdk/kan cassette produced by BsmBI digestion.

    Characterization

    (DATA HERE!)

    [1] Metzgar, D., Bacher, J. M., Pezo, V., Reader, J., Doring, V., Schimmel, P., Marliere, P., & de Crecy-Lagard, V. (2004). Acinetobacter sp.. ADP1: An ideal model organism for genetic analysis and Genome Engineering. Nucleic Acids Research, 32(19), 5780–5790. https://doi.org/10.1093/nar/gkh881.

    [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.