View source for Part:BBa K4342015


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<h1>Introduction</h1> 

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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>

The <em> nptII </em> gene in <em> Acinetobacter baylyi </em> ADP1 codes for kanamycin antibiotic resistance. Deleting a section of this gene prevents ADP1 from growing on kanamycin antibiotic, making ADP1 susceptible to this antibiotic. This allows for the detection of homologous recombination/transformation of the correct <em> nptII </em> antibiotic resistance gene by AdDP1.

<h1>Design</h1>

The <em> nptII </em> broken gene part comprises the 1583 base pair region that contains the deactivated <em> nptII </em> gene in ADP1. This part has BsaI restriction sites attached to the 5’ end and the 3’ end, which are specifically designed to allow for the detection of a functional <em> nptII </em> gene through a two-step process involving selection and counterselection.

===BsaI Restriction Site===
The <b>BsaI site</b> is designed to ligate to the 3’ end of the <em> ACIAD2049 </em> Upstream [https://parts.igem.org/Part:BBa_K4342001 (BBa_4342001)] and the 5' end of the <em> ACIAD2049 </em> Downstream [https://parts.igem.org/Part:BBa_K4342002 (BBa_4342002)] creating the <em> nptII </em> detector construct composite part [https://parts.igem.org/Part:BBa_K4342031 (BBa_4342031)]. This composite part permits the selection of transformants through kanamycin resistance.

<h1>References</h1>
[1] De Vries, J., Wackernagel, W. (1998). Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation. Molecular and General Genetics, 257, 606-613. https://doi.org/10.1007/s004380050688

[2] Kong, K., Schneper, L., Mathee, K. (2010). Beta-lactam Antibiotics: From Antibiosis to Resistance and Bacteriology. APMIS, 118(1), 1-36. https://doi.org/10.1111/j.1600-0463.2009.02463.x


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