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		+	__NOTOC__
	<partinfo>BBa_K4342010 short</partinfo>		<partinfo>BBa_K4342010 short</partinfo>
−	===Usage and Biology===	+	<h1>Introduction</h1>
		+
		+	[[File:intro-part-figure.png|500px|thumb|right|]]
		+
		+
		+
		+	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>
		+
		+
		+
		+
		+	<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 Downstream</i></b> is categorized as a Type 5 <b> Downstream </b> basic part in our part collection.
		+
		+
		+
		+	<h1>Usage and Biology</h1>
		+
		+	<i>acrB</i> is a gene in <em> Acinetobacter baylyi </em> ADP1 which codes for proteins involved with efflux pumps [1]. <i>acrB</i> also contributes to intrinsic 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, we have inserted a mutated <i> TEM-1 </i> gene [https://parts.igem.org/Part:BBa_K4342017 (BBa_4342017)] in place of <i>acrB</i> to detect the presence of a Wild-Type <i>TEM-1</i> gene, showing how ADP1 can be engineered to detect antibiotic resistance.
		+
		+	<h1>Design</h1>
		+
		+	The <b><i>acrB</i> Downstream</b> part comprises the 1027 bp homology directly downstream of the <i>acrB</i> gene in ADP1.
		+	We designed optimized primers, which include GC contents of over 40% and melting temperatures of under 70 °C. BsaI and BsmBI restriction sites are attached to the 5’ end, which are designed to ligate to the 3' end of the <i>tdk/kan</i> cassette [https://parts.igem.org/Part:BBa_K4342000 (BBa_4342000)] and the <i>acrB</i> Upstream part [https://parts.igem.org/Part:BBa_K4342009 (BBa_4342009)] respectively. 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.
		+
		+	This part contains a BsaI restriction site with a standard 4 bp GGA Type 2 Prefix [2] and a BsmBI restriction site with a 4 bp “rescue” complementary scar. See the [https://2022.igem.wiki/austin-utexas/contribution Contribution] page on our wiki 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 is used to assemble the <i>acrB</i> integration cassette [https://parts.igem.org/Part:BBa_K4342023 (BBa_4342023)] and the <i>acrB</i> rescue cassette [https://parts.igem.org/Part:BBa_K4342024 (BBa_4342024)] composite parts. Figures 1 and 2 show how these composite parts can be used in our two-step ADP1 Genetic Engineering protocol to create a minimal 4 bp scar in the deletion of the <i>acrB</i> gene.
		+
		+	===Step 1===
		+	This part is designed to ligate to the 3' end of the <em> tdk/kan </em> cassette, [https://parts.igem.org/Part:BBa_K4342000 BBa_4342000], creating the <i>acrB</i>-<em> tdk/kan </em> cassette composite part [https://parts.igem.org/Part:BBa_K4342023 (BBa_4342023)]. 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|<b> Fig. 1. </b> The insertion of the <i>tdk/kan</i> cassette in place of a target gene (<i>acrB</i>).]]
		+	===Step 2===
		+	The <i>tdk/kan</i> cassette can subsequently be knocked out to create a deletion of <i>acrB</i> via BsmBI digestion, [https://parts.igem.org/Part:BBa_K4342020 BBa_4342020], with a 4 bp minimal scar. During this reaction, this part is ligated to the 5' end of the <i>acrB</i> Upstream part [https://parts.igem.org/Part:BBa_K4342009 (BBa_4342009)]. 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|<b> Fig. 2. </b> The deletion of the <i>tdk/kan</i> cassette produced by BsmBI digestion.]]
		+
		+	<h1>Characterization</h1>
		+
		+	[[File:tdkkan1.png|500px|thumb|center|<b> Fig. 3. </b> Growth on kanamycin plates indicate the successful integration of the <i>tdk/kan</i> cassette into ADP1's genome.]]
		+
		+	[[File:tdkkan2.png|500px|thumb|center|<b> Fig. 4. </b> Growth on AZT plates indicate the successful removal of the <i>tdk/kan</i> cassette from ADP1's genome.]]
		+
		+
		+	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 ~1000 bp, confirming the amplification of the <b><i>acrB</i> Upstream</b> part. A PCR master mix with diH<sub>2</sub>O in place of template DNA was used as negative control. Additionally, we performed a PCR to confirm the replacement of the <i>acrB</i> gene with the <i>tdk/kan</i> cassette. (Fig. 3).
		+
		+	[[File:AcrB_Deletion_Gel.png|500px|thumb|center|<b> Fig. 5. </b> PCR showing the successful deletion of the <i>acrB</i> gene, using the <i>tdk/kan</i> part.]]
		+
−	The AcrB gene participates in the efflux of β-lactam antibiotics such as ampicillin and carbenicillin. The efflux process makes ADP1 more resistant to these antibiotics. Deleting the acrB gene increases ADP1's susceptibility to ampicillin, carbenicillin, and other β-lactams. Additionally, the AcrB gene is non-essential for ADP1’s survival, thus this gene serves as an ideal location for inserting genetic constructs.	+	<h1>References</h1>
−	The acrB downstream homology part comprises the 1011 base pair region directly downstream of the acrB gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 5’ end. These restriction sites are designed to delete the acrB gene through a two-step process involving selection and counterselection.	+	[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
−	===BsaI Restriction Site===	+	[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.
−	The bsaI site is designed to ligate to the 5’ end of the tdk/kan cassette (BBa) creating the AcrB tdk/kan cassette composite part (BBa). This composite part permits the selection of transformants through kanamycin resistance.	+
−	===Bsmbi Restriction Site===	+	<partinfo>BBa_K4342010 SequenceAndFeatures</partinfo>
−	Designed to ligate to the 3’ end of the AcrB Upstream homology (BBa) creating the ΔAcrB homologies composite part (BBa). This composite part permits the counterselection of  transformants when plating on Azidothymidine (AZT).	+

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Latest revision as of 03:19, 14 October 2022

acrB Downstream

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 Downstream is categorized as a Type 5 Downstream basic part in our part collection.

Usage and Biology

acrB is a gene in Acinetobacter baylyi ADP1 which codes for proteins involved with efflux pumps [1]. acrB also contributes to intrinsic 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, we have inserted a mutated TEM-1 gene (BBa_4342017) in place of acrB to detect the presence of a Wild-Type TEM-1 gene, showing how ADP1 can be engineered to detect antibiotic resistance.

Design

The acrB Downstream part comprises the 1027 bp homology directly downstream of the acrB gene in ADP1. We designed optimized primers, which include GC contents of over 40% and melting temperatures of under 70 °C. BsaI and BsmBI restriction sites are attached to the 5’ end, which are designed to ligate to the 3' end of the tdk/kan cassette (BBa_4342000) and the acrB Upstream part (BBa_4342009) respectively. See Figure 4 on the Engineering Page for more details on how to design primers containing the correct GGA Type Overhang and restriction sites.

This part contains a BsaI restriction site with a standard 4 bp GGA Type 2 Prefix [2] and a BsmBI restriction site with a 4 bp “rescue” complementary scar. See the Contribution page on our wiki 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 is used to assemble the acrB integration cassette (BBa_4342023) and the acrB rescue cassette (BBa_4342024) composite parts. Figures 1 and 2 show how these composite parts can be used in our two-step ADP1 Genetic Engineering protocol to create a minimal 4 bp scar in the deletion of the acrB gene.

Step 1

This part is designed to ligate to the 3' end of the tdk/kan cassette, BBa_4342000, creating the acrB- tdk/kan cassette composite part (BBa_4342023). 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 deletion of acrB via BsmBI digestion, BBa_4342020, with a 4 bp minimal scar. During this reaction, this part is ligated to the 5' end of the acrB Upstream part (BBa_4342009). 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 ~1000 bp, confirming the amplification of the acrB Upstream part. A PCR master mix with diH₂O in place of template DNA was used as negative control. Additionally, we performed a PCR to confirm the replacement of the acrB gene with the tdk/kan cassette. (Fig. 3).

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.