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−	<partinfo>BBa_K4342013 short</partinfo>
−	<h1>Usage and Biology</h1>	+	__NOTOC__
		+	<partinfo>BBa_K4342012 short</partinfo>
−	The pbpG gene codes for a penicillin-binding protein that increases ADP1’s resistance to β-lactam antibiotics. Thus deleting the pbpG gene makes ADP1 more susceptible to ampicillin, carbenicillin, and other β-lactams. Additionally, the pbpG gene is non-essential for ADP1’s survival and serves as an ideal location for inserting genetic constructs.	+	<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)].
		+
		+
		+
		+	<h1>Usage and Biology</h1>
		+
		+	<i>pbpG</i> is a nonessential gene in <em> Acinetobacter baylyi </em> ADP1 that codes for a protein involved in peptidoglycan synthesis, contributing 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>pbpG</i> can be replaced with any DNA construct.
	<h1>Design</h1>		<h1>Design</h1>
−	The pbpG downstream homology part comprises the 1019 base pair region directly upstream of the pbpG gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 5’ end which are designed to delete pbpG through a two-step process involving selection and counterselection.	+	The <b><i>pbpG</i> Downstream </b> part comprises the 977 bp homology directly downstream of the <i>pbpG</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>pbpG</i> Upstream part [https://parts.igem.org/Part:BBa_K4342011 (BBa_4342011)] 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.
−	===BsaI Restriction Site===	+	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.
−	The <b>bsaI site</b> is designed to ligate to the 3’ end of the tdk/kan cassette [https://parts.igem.org/Part:BBa_K4342000 (BBa_K4342000)] creating the pbpG tdk/kan cassette composite part (BBa). This composite part permits the selection of transformants through kanamycin resistance.	+
−	===Bsmbi Restriction Site===	+	==Composite Parts==
−	The <b>bsmbI site</b> is designed to ligate to the 5’ end of the pbpG upstream homology [https://parts.igem.org/Part:BBa_K4342011 (BBa_K4342011)] creating the ΔpbpG homologies composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT).	+	This basic part is used to assemble the <i>pbpG</i> integration cassette [https://parts.igem.org/Part:BBa_K4342021 (BBa_4342021)] and the <i>pbpG</i> rescue cassette [https://parts.igem.org/Part:BBa_K4342022 (BBa_4342022)]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>pbpG</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>pbpG</i>-<em> tdk/kan </em> cassette composite part [https://parts.igem.org/Part:BBa_K4342021 (BBa_4342021)]. 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>pbpG</i>).]]
		+	===Step 2===
		+	The <i>tdk/kan</i> cassette can subsequently be knocked out to create a deletion of <i>pbpG</i> via BsmBI digestion, [https://parts.igem.org/Part:BBa_K4342022 BBa_4342022], with a minimal 4 bp scar. During this reaction, this part is ligated to the 3' end of the <i>pbpG</i> Upstream part [https://parts.igem.org/Part:BBa_K4342011 BBa_4342011]. 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>		<h1>Characterization</h1>
−	<h1>References</h1>	+	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>pbpG</i> Downstream</b> part. A PCR master mix with diH<sub>2</sub>O in place of template DNA was used as negative control.
		+	[[File:pbpG synthesis.png|500px|thumb|center|<b> Fig. 3. Synthesis of <i>pbpG</i> Homology Flanks and <i>tdk/kan</i> Transformation Cassettes. (A)</b> PCR Amplification of <i>pbpG</i> 5’ Upstream flanks (1 kb) and 3’ Downstream flanks using an annealing temperature gradient of 53°C to 60°C. A faint band in the upstream negative control is likely due to leaky contamination. (B) BsaI GGA of <i>pbpG</i> Upstream and Downstream flanks with pBTK622, containing the <i>tdk/kan</i> cassette. Lane 1: 3.7 kb <i>tdk/kan</i> ligated to homology flanks.]]
−	<!-- Add more about the biology of this part here	+	[[File:tdkkan1.png|500px|thumb|center|<b> Fig. 4. </b> Growth on kanamycin plates indicate the successful integration of the <i>tdk/kan</i> cassette into ADP1's genome.]]
−	===Usage and Biology===	+
−	<!-- -->
−	<span class='h3bb'>Sequence and Features</span>
−	<partinfo>BBa_K4342012 SequenceAndFeatures</partinfo>
		+	[[File:tdkkan2.png|500px|thumb|center|<b> Fig. 5. </b> Growth on AZT plates indicate the successful removal of the <i>tdk/kan</i> cassette from ADP1's genome.]]
−	<!-- Uncomment this to enable Functional Parameter display	+
−	===Functional Parameters===	+	[[File:pbpG deletion.png|500px|thumb|center|<b> Fig. 6. PCR Confirmation of the <i>pbpG</i> Deletion from ADP1.</b>]]
−	<partinfo>BBa_K4342012 parameters</partinfo>	+
−	<!-- -->	+
		+
		+	<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. ACS synthetic biology 4, 975–986. 10.1021/sb500366v.
		+
		+
		+	<partinfo>BBa_K4342012 SequenceAndFeatures</partinfo>

---

Latest revision as of 03:41, 14 October 2022

pbpG Downstream Homology

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).

Usage and Biology

pbpG is a nonessential gene in Acinetobacter baylyi ADP1 that codes for a protein involved in peptidoglycan synthesis, contributing 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 pbpG can be replaced with any DNA construct.

Design

The pbpG Downstream part comprises the 977 bp homology directly downstream of the pbpG 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 pbpG Upstream part (BBa_4342011) 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 pbpG integration cassette (BBa_4342021) and the pbpG rescue cassette (BBa_4342022)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 pbpG gene.

Step 1

This part is designed to ligate to the 3' end of the tdk/kan cassette, BBa_4342000, creating the pbpG- tdk/kan cassette composite part (BBa_4342021). 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 pbpG via BsmBI digestion, BBa_4342022, with a minimal 4 bp scar. During this reaction, this part is ligated to the 3' end of the pbpG Upstream part BBa_4342011. 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 pbpG Downstream part. A PCR master mix with diH₂O in place of template DNA was used as negative control.

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.