View source for Part:BBa K4342008


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<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>cymR</i> YFP</b> is categorized as a Type 2-4 <b>Genetic Device</b> basic part in our part collection.


<h1>Usage and Biology</h1>
The <em> CymR </em> YFP in <em> Acinetobacter baylyi </em> ADP1 codes for a yellow fluorescent protein.

<h1>Design</h1>
The <em> CymR </em> YFP part comprises the 1014 base pair region in place of the <i>acrB</i> gene of ADP1. This part has BsaI restriction sites attached to the 5’ end and 3’ ends, which are specifically designed to ligate to the acrB Upstream Homology [https://parts.igem.org/Part:BBa_K4342009 (BBa_4342009)] and the acrB Downstream Homology [https://parts.igem.org/Part:BBa_K4342010 (BBa_4342010)]via BsaI digestion in the <em> P. destructans </em> Detector strain.

===BsaI Restriction Sites===
The <b>BsaI sites</b> are designed to ligate to the 3’ end of the <em> acrB </em> Upstream Homology [https://parts.igem.org/Part:BBa_K4342009 (BBa_4342009)] and the 5’ end of the <em> acrB </em> Downstream Homology [https://parts.igem.org/Part:BBa_K4342010 (BBa_4342010)] This basic part permits the expression of a YFP gene using this promoter, which allows for screenable selection when the <em> CymR </em> repressor (BBa_4342007) is deleted through homologous recombination of <em> P. destructans </em> eDNA.

<h1>Characterization</h1>
[[File:Yfp-insertion-gel.png|500px|thumb|center|<b> Fig. 3. YFP Insertions. From left to right: Ladder, Control, ADP1-ISx, <i>cymR</i>-YFP, <i>vanR</i>-YFP, <i>lacI</i>-YFP, and <i>betI</i>-YFP.</b> Expected length for each YFP variation was ~3000 base pairs.]]

<h1>References</h1>
[1] Meyer, A., Shegall-Shapiro, T., Glassey, E., Zhang, J., Voigt, C. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small molecule sensors. Nature Chemical Biology 15, 196-204 (2019). https://doi.org/10.1038/s41589-018-0168-3

<partinfo>BBa_K4342008 SequenceAndFeatures</partinfo>