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	<partinfo>BBa_K4342024 short</partinfo>		<partinfo>BBa_K4342024 short</partinfo>
		+	<h1>Introduction</h1>
		+	[[File:intro-part-figure.png|500px|thumb|right|]]
−	<!-- Add more about the biology of this part here
−	===Usage and Biology===
−	<!-- -->	+	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.
−	<span class='h3bb'>Sequence and Features</span>	+
−	<partinfo>BBa_K4342024 SequenceAndFeatures</partinfo>	+
		+	<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>
−	<!-- Uncomment this to enable Functional Parameter display	+
−	===Functional Parameters===	+
−	<partinfo>BBa_K4342024 parameters</partinfo>	+
−	<!-- -->	+	<h1>Categorization</h1>
		+	For our parts collection, we categorize our parts into the following categories:
		+
		+	<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)].
		+
		+
		+	<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)].
		+
		+
		+	<b> Rescue Cassettes </b>
		+
		+	<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).
		+
		+
		+	<b> Genetic Device </b>
		+
		+	<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)].
		+
		+
		+	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)].
		+
		+
		+
		+
		+	<b> <i>acrB</i> Rescue </b> is categorized as a <b> Rescue </b> cassette in our part collection. This part is not categorized by a GGA Type Overhang because this part is ligated via BsmBI digestion, rather than BsaI digestion.
		+
		+	<h1>Usage and Biology</h1>
		+
		+	<i>acrB</i> is a gene in <em> Acinetobacter baylyi </em> ADP1 that encodes proteins involved with efflux pumps [1]. It also contributes to β-lactam antibiotic resistance[1]. Knocking out this gene allows for the integration of other DNA sequences in its chromosomal location. Using this part, we demonstrate that <i>acrB</i> can be replaced with any DNA construct. Specifically, the <b>ACIAD2049 Rescue</b> cassette knocks out <i>acrB</i> from ADP1.
		+
		+	<h1>Design</h1>
		+
		+	The <b><i>acrB</i> Rescue</b> part comprises the 2047 bp region combining the <i>acrB</i> Upstream [https://parts.igem.org/Part:BBa_K4342009 (BBa_4342009)] and <i>acrB</i> Downstream [https://parts.igem.org/Part:BBa_K4342010 (BBa_4342010)] parts.
		+
		+	*Please note that BsaI restriction sites have been removed to meet RFC[1000] BioBrick Assembly Compatibility. To see in-depth primer design, please see Figure 4 on the [https://2022.igem.wiki/austin-utexas/engineering Engineering Page] for more details on how to design primers containing the correct GGA Type Overhang and restriction sites.
		+
		+	===Step 1===
		+	The <b><i>acrB</i> Integration</b> cassette [https://parts.igem.org/Part:BBa_K4342023 (BBa_4342023)] is designed to allow for successful transformant selection on Kanamycin (Kan) via the <i>kanR</i> gene (Figure 1).
		+	[[File:TdkKan_Selection.png|500px|thumb|center|<b> Fig. 1. </b> First-Step Integration of the <i>tdk/kan</i> cassette in place of an ADP1 Target Gene (<i>acrB</i>).]]
		+	===Step 2===
		+	The <i>tdk/kan</i> cassette can subsequently be knocked out to create a 4 bp minimal scar deletion of <i>acrB</i> via BsmBI digestion. During this second-step transformation, we transform this <b><i>acrB</i> Rescue</b> cassette into ADP1 containing the <b><i>acrB</i> Integration</b> cassette [https://parts.igem.org/Part:BBa_K4342023 (BBa_4342023)]. Using this part allows us to select for successful transformants on Azidothymidine (AZT) (Figure 2). Successful transformants have removed the <i>tdk/kan</i> cassette and integrated our <b><i>acrB</i> Rescue</b> cassette, resulting in the deletion of <i>acrB</i> from ADP1. [[File:TdkKan_Counterselection.png|500px|thumb|center|<b> Fig. 2. </b> Second-Step Removal of the <i>tdk/kan</i> cassette.]]
		+
		+
		+	<h1>Characterization</h1>
		+	To confirm that we successfully created this part, we transformed the <b><i>acrB</i> Rescue</b> cassette into ADP1-ISx [3] containing the <b><i>acrB</i> Integration</b> cassette. We selected for successful transformants on LB-AZT plates and picked colonies to inoculate and store frozen stocks of our newly constructed deletion strain.
		+
		+	<h1>References</h1>
		+
		+	[1] Gomez, M. J., & Neyfakh, A. A. (2006). Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrobial agents and chemotherapy, 50(11), 3562-3567. https://doi.org/10.1128/AAC.00579-06
		+
		+	[2] Lee, M.E., DeLoache, W.C., Cervantes, B., and Dueber, J.E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. <i>ACS synthetic biology</i> 4, 975–986. 10.1021/sb500366v.
		+
		+	[3] Suárez, G. A., Renda, B. A., Dasgupta, A., & Barrick, J. E. (2017). Reduced Mutation Rate and Increased Transformability of Transposon-Free <i>Acinetobacter baylyi</i> ADP1-ISx. <i>Applied and environmental microbiology</i>, 83(17), e01025-17. https://doi.org/10.1128/AEM.01025-17
		+
		+	<partinfo>BBa_K4342024 SequenceAndFeatures</partinfo>

---

Latest revision as of 01:58, 14 October 2022

acrB Rescue 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

"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

"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).

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).

acrB Rescue is categorized as a Rescue cassette in our part collection. This part is not categorized by a GGA Type Overhang because this part is ligated via BsmBI digestion, rather than BsaI digestion.

Usage and Biology

acrB is a gene in Acinetobacter baylyi ADP1 that encodes proteins involved with efflux pumps [1]. It also contributes to β-lactam antibiotic resistance[1]. Knocking out this gene allows for the integration of other DNA sequences in its chromosomal location. Using this part, we demonstrate that acrB can be replaced with any DNA construct. Specifically, the ACIAD2049 Rescue cassette knocks out acrB from ADP1.

Design

The acrB Rescue part comprises the 2047 bp region combining the acrB Upstream (BBa_4342009) and acrB Downstream (BBa_4342010) parts.

• Please note that BsaI restriction sites have been removed to meet RFC[1000] BioBrick Assembly Compatibility. To see in-depth primer design, please see Figure 4 on the Engineering Page for more details on how to design primers containing the correct GGA Type Overhang and restriction sites.

Step 1

The acrB Integration cassette (BBa_4342023) is designed to allow for successful transformant selection on Kanamycin (Kan) via the kanR gene (Figure 1).

Step 2

The tdk/kan cassette can subsequently be knocked out to create a 4 bp minimal scar deletion of acrB via BsmBI digestion. During this second-step transformation, we transform this acrB Rescue cassette into ADP1 containing the acrB Integration cassette (BBa_4342023). Using this part allows us to select for successful transformants on Azidothymidine (AZT) (Figure 2). Successful transformants have removed the tdk/kan cassette and integrated our acrB Rescue cassette, resulting in the deletion of acrB from ADP1.

Characterization

To confirm that we successfully created this part, we transformed the acrB Rescue cassette into ADP1-ISx [3] containing the acrB Integration cassette. We selected for successful transformants on LB-AZT plates and picked colonies to inoculate and store frozen stocks of our newly constructed deletion strain.

References

[1] Gomez, M. J., & Neyfakh, A. A. (2006). Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrobial agents and chemotherapy, 50(11), 3562-3567. https://doi.org/10.1128/AAC.00579-06

[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.

[3] Suárez, G. A., Renda, B. A., Dasgupta, A., & Barrick, J. E. (2017). Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx. Applied and environmental microbiology, 83(17), e01025-17. https://doi.org/10.1128/AEM.01025-17