iGEM 2022 — Team austin-utexas

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# Notebook

# Weekly Lab Notebooks

# March

[Week 1 (3/26/22 - 3/27/22)](#1)

# April

[Week 2 (4/2/22 -4/3/22)](#2)
[Week 3 (4/9/22 - 4/13/22)](#3)
[Week 4 (4/16/22 - 4/18/22)](#4)
[Week 5 (4/23/22 - 4/24/22)](#5)

# May

[Week 6 (5/19/22 - 5/26/22)](#6)

# June

[Week 7 (6/1/22 - 6/5/22)](#7)
[Week 8 (6/6/22 - 6/10/22)](#8)
[Week 9 (6/11/22-6/18/22)](#9)
[Week 10 (6/19/22 - 6/25/22)](#10)
[Week 11 (6/26/22 -7/2/22)](#11)

# July

[Week 12 (7/3/22 - 7/9/22)](#12)
[Week 13 (7/10/22 - 7/16/22)](#13)
[Week 14 (7/17/22 - 7/23/22)](#14)
[Week 15 (7/24/22 - 7/30/22)](#15)

# August

[Week 16 (8/1/22 - 8/7/22)](#16)
[Week 17 (8/16/22 - 8/21/22)](#17)
[Week 18 (8/22/22 - 9/1/22)](#18)

# September

[Week 19 (9/3/22 - 9/10/22)](#19)
[Week 20 (9/11/22 - 9/18/22)](#20)
[Week 21 (9/19/22 - 9/25/22)](#21)
[Week 22 (9/16/22 - 10/1/22)](#22)

# October

[Week 23 (10/4/22 - 10/9/22)](#23)

# Week 1 (3/26/22 - 3/27/22)

[Keaton's Notebook 3/26](https://static.igem.wiki/teams/4342/wiki/notebook/3-26-keaton-brown-benchling.pdf)

[Keaton's Notebook 3/27](https://static.igem.wiki/teams/4342/wiki/notebook/3-27-28-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 3/26](https://static.igem.wiki/teams/4342/wiki/notebook/3-26-27-jeffrey-chuong-benchling.pdf)
[Leia's Notebook 3/26](https://static.igem.wiki/teams/4342/wiki/notebook/3-26-27-leia-jiang-benchling.pdf)

# Week 2 (4/2/22 - 4/3/22)

[Jeffrey's Notebook 4/2](https://static.igem.wiki/teams/4342/wiki/notebook/4-2-3-jeffrey-chuong-benchling.pdf)
[Leia's Notebook 4/2](https://static.igem.wiki/teams/4342/wiki/notebook/4-2-3-leia-jiang-benchling.pdf)

# Week 3 (4/9/22 - 4/13/22)

[Keaton's Notebook 4/8](https://static.igem.wiki/teams/4342/wiki/notebook/4-8-12-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 4/9](https://static.igem.wiki/teams/4342/wiki/notebook/4-9-13-jeffrey-chuong-benchling.pdf)

# Week 4 (4/16/22 - 4/18/24)

[Keaton's Notebook 4/16](https://static.igem.wiki/teams/4342/wiki/notebook/4-16-24-keaton-brown-benchling.pdf)

[Keaton's Notebook 4/23](https://static.igem.wiki/teams/4342/wiki/notebook/4-23-24-keaton-brown-benchling.pdf)
[Jeffrey's Notebook 4/16](https://static.igem.wiki/teams/4342/wiki/notebook/4-16-17-jeffrey-chuong-benchling.pdf)
[Leia's Notebook 4/16](https://static.igem.wiki/teams/4342/wiki/notebook/4-16-18-leia-jiang-benchling.pdf)

# Week 5 (4/23/22 - 4/24/22)

[Keaton's Notebook 4/23](https://static.igem.wiki/teams/4342/wiki/notebook/4-23-24-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 4/23](https://static.igem.wiki/teams/4342/wiki/notebook/4-23-24-jeffrey-chuong-benchling.pdf)

[Leia's Notebook 4/23](https://static.igem.wiki/teams/4342/wiki/notebook/4-23-24-leia-jiang-benchling.pdf)

# Week 6 (5/19/22 - 5/26/22)

[Keaton's Notebook 5/19](https://static.igem.wiki/teams/4342/wiki/notebook/5-19-24-keaton-brown-benchling.pdf)

[Keaton's Notebook 5/26](https://static.igem.wiki/teams/4342/wiki/notebook/5-26-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 5/20](https://static.igem.wiki/teams/4342/wiki/notebook/5-20-jeffrey-chuong-benchling.pdf)

# Week 7 (6/1/22 - 6/5/22)

[Keaton's Notebook 6/1](https://static.igem.wiki/teams/4342/wiki/notebook/6-1-week-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 6/1](https://static.igem.wiki/teams/4342/wiki/notebook/6-1-5-jeffrey-chuong-benchling.pdf)

# Week 8 (6/6/22 - 6/10/22)

[Keaton's Notebook 6/6](https://static.igem.wiki/teams/4342/wiki/notebook/6-6-week-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 6/6](https://static.igem.wiki/teams/4342/wiki/notebook/6-6-12-jeffrey-chuong-benchling.pdf)

[Leia's Notebook 6/8](https://static.igem.wiki/teams/4342/wiki/notebook/6-8-10-leia-jiang-benchling.pdf)

# Week 9 (6/11/22 - 6/18/22)

[Keaton's Notebook 6/13](https://static.igem.wiki/teams/4342/wiki/notebook/6-13-week-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 6/13](https://static.igem.wiki/teams/4342/wiki/notebook/6-13-19-jeffrey-chuong-benchling.pdf)

# Week 10 (6/19/22 - 6/25/22)

[Keaton's Notebook 6/20](https://static.igem.wiki/teams/4342/wiki/notebook/6-20-week-keaton-brown-benchling.pdf)

[Leia's Notebook 6/20](https://static.igem.wiki/teams/4342/wiki/notebook/6-20-24-leia-jiang-benchling.pdf)

# Week 11 (6/26/22-7/2/22)

[Jeffrey's Notebook 6/27](https://static.igem.wiki/teams/4342/wiki/notebook/6-27-7-4-jeffrey-chuong-benchling.pdf)

[Leia's Notebook 6/28](https://static.igem.wiki/teams/4342/wiki/notebook/6-28-30-leia-jiang-benchling.pdf)

# Week 12 (7/3/22 - 7/9/22)

[Keaton's Notebook 7/6](https://static.igem.wiki/teams/4342/wiki/notebook/7-6-week-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 7/5](https://static.igem.wiki/teams/4342/wiki/notebook/7-5-10-jeffrey-chuong-benchling.pdf)

[Leia's Notebook 7/5](https://static.igem.wiki/teams/4342/wiki/notebook/7-5-10-leia-jiang-benchling.pdf)

# Week 13 (7/10/22 - 7/16/22)

[Keaton's Notebook 7/11](https://static.igem.wiki/teams/4342/wiki/notebook/7-11-week-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 7/11](https://static.igem.wiki/teams/4342/wiki/notebook/7-11-17-jeffrey-chuong-benchling.pdf)

[Leia's Notebook 7/11](https://static.igem.wiki/teams/4342/wiki/notebook/7-11-15-leia-jiang-benchling.pdf)

# Week 14 (7/17/22 - 7/23/22)

[Keaton's Notebook 7/20](https://static.igem.wiki/teams/4342/wiki/notebook/7-20-week-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 7/18](https://static.igem.wiki/teams/4342/wiki/notebook/7-18-24-jeffrey-chuong-benchling.pdf)

[Leia's Notebook 7/19](https://static.igem.wiki/teams/4342/wiki/notebook/7-19-22-leia-jiang-benchling.pdf)

# Week 15 (7/24/22 - 7/30/22)

[Leia's Notebook 7/28](https://static.igem.wiki/teams/4342/wiki/notebook/7-28-leia-jiang-benchling.pdf)

# Week 16 (8/1/22 - 8/8/22)

[Keaton's Notebook 8/1](https://static.igem.wiki/teams/4342/wiki/notebook/8-1-week-keaton-brown-benchling.pdf)

[Keaton's Notebook 8/8](https://static.igem.wiki/teams/4342/wiki/notebook/8-8-week-keaton-brown-benchling.pdf)
[Leia's Notebook 8/8](https://static.igem.wiki/teams/4342/wiki/notebook/8-8-leia-jiang-benchling.pdf)

# Week 17 (8/16/22 - 8/21/22)

[Leia's Notebook 8/16](https://static.igem.wiki/teams/4342/wiki/notebook/8-16-21-leia-jiang-benchling.pdf)

# Week 18 (8/22/22 - 9/1/2022)

[Keaton's Notebook 8/22](https://static.igem.wiki/teams/4342/wiki/notebook/8-22-week-keaton-brown-benchling.pdf)

[Jeffrey's Notebook 8/22](https://static.igem.wiki/teams/4342/wiki/notebook/igem-8-22-jeffrey.pdf)

Leia's Notebook 8/22

# Week 19 (9/3/22 - 9/10/22)

[Jeffrey's Notebook 9/3](https://static.igem.wiki/teams/4342/wiki/notebook/igem-9-3-10-jeffrey-chuong-benchling.pdf)

# Week 20 (9/11/22 - 9/18/22)

[Jeffrey's Notebook 9/11](https://static.igem.wiki/teams/4342/wiki/notebook/igem-9-11-18-jeffrey-chuong-benchling.pdf)

# Week 21 (9/19/22 - 9/25/22)

[Jeffrey's Notebook 9/19](https://static.igem.wiki/teams/4342/wiki/notebook/igem-9-19-25-jeffrey-chuong-benchling.pdf)

# Week 22 (9/16/22 - 10/1/22)

[Jeffrey's Notebook 9/19](https://static.igem.wiki/teams/4342/wiki/notebook/igem-9-19-25-jeffrey-chuong-benchling.pdf)

# Week 23 (10/4/22 - 10/9/22)

[Jeffrey's Notebook 10/4](https://static.igem.wiki/teams/4342/wiki/notebook/jeffrey-chuong-10-4-10-12-benchling.pdf)

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####

© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)
The repository used to create this website is available at [gitlab.igem.org/2022/austin-utexas.](https://gitlab.igem.org/2022/austin-utexas)

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# Engineering

# Engineering

[• Introduction](#intro)
[• Phase 1: *ampD* Engineering](#p1)
[• Phase 2: *pbpG* and *acrB* Engineering](#p2)
[• Phase 3: *recJ* and ACIAD2049 Engineering](#p3)

![](https://static.igem.wiki/teams/4342/wiki/design-cycle.png)

**Fig. 1. Design-Build-Test-Learn Cycle for ADP1 Genetic Engineering.**

# Introduction

*Acinetobacter baylyi* ADP1 is a bacterium capable of taking up DNA directly from the environment (Figure 2). Despite its natural transformation abilities, it is not as well-documented or as commonly used as a synthetic biology chassis as *E. coli*. We used a reliable, two-step protocol, as described by [1], to genetically engineer ADP1. **Throughout the 2022 iGEM season, we went through multiple iterations of the Design-Build-Test-Learn process when making changes to ADP1’s genome.**

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-2.png)

**Fig. 2. *Acinetobacter baylyi* ADP1's Natural Competence Allows for Uptake of DNA, such as PCR product or Golden Gate Assembly DNA.** Adapted from [1].

Here, we describe the phases of our engineering process, using ADP1’s homologous recombination mechanism to our advantage (Figure 3). In each phase, we use standard molecular cloning techniques, including **Polymerase Chain Reaction (PCR)** and **Golden Gate Assembly (GGA)**, to amplify and construct our DNA sequences. We demonstrate simple confirmation methods using the *tdk/kan* cassette with selective antibiotic agar plates for bacterial growth. Lastly, we explain strategies used to troubleshoot problems we ran into while following our Design-Build-Test-Learn cycles.

![](https://static.igem.wiki/teams/4342/wiki/screenshot-2022-10-06-212449.png)

**Fig. 3. *Acinetobacter baylyi* ADP1 Homologous Recombination Mechanism used for Genetically Modifying ADP1.** The ADP1 chromosome is combined with a transformant DNA fragment that contains matching endogenous ADP1 gene upstream and downstream sequences. The left side shows transformant DNA that has a foreign DNA sequence in between gene homology flanks, demonstrating the integration of foreign DNA into ADP1’s chromosome. The right side shows transformant DNA as simply the gene homology flanks ligated together, demonstrating a gene deletion.

# Phase 1: *ampD* Engineering

We chose to knock out *ampD*, an ADP1 gene that contributes to intrinsic β-lactam antibiotic resistance, to improve ADP1’s usability as a synthetic biology chassis. *ampD* is involved in breaking down peptidoglycan proteins [3]. Although we failed to knock out *ampD*, we applied what we learned to our future ADP1 genetic engineering phases.

# Design

We designed primers to amplify ~1 kb homology 5’ and 3’ flanks upstream and downstream of the endogenous ADP1 target gene (Figure 4). Each set of primers contains a “Golden” primer that attaches BsaI and BsmBI restriction sites to the end of the primer, containing the correct GGA Type Overhang (BsaI) and 4 bp "rescue" complementary scar (BsmBI). See the [Contribution Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D) for more details on how we utilize flexible GGA Type Overhangs to build our system. These amplified homology flanks, once assembled, are used to modify the ADP1 genome. In the first transformation step, the upstream and downstream flanks are ligated with the *tdk/kan* cassette using GGA via BsaI digestion. In the second transformation step, the upstream and downstream flanks are ligated to each other using GGA via BsmBI digestion.

![](https://static.igem.wiki/teams/4342/wiki/primer-design-1.png)

**Fig. 4. Primer Design for ADP1 Genetic Engineering.** Designed primers amplify the endogenous ADP1 target gene's upstream and downstream region to create homology flanks that will be used in later steps. Primers contain BsaI and BsmBI restriction sites for GGA. We used the NEB Tm Calculator to determine primer annealing temperatures.

# Build

We synthesized homology flanks using PCR using our designed *ampD* primers and ADP1-ISx genomic DNA. We used gel electrophoresis to verify 1 kb upstream and downstream flank fragment sizes (Figure 5A, B) and the Zymo DNA Clean and Concentrate Kit to purify PCR products.

Next, we used GGA to ligate the upstream and downstream homology flanks with the *tdk/kan* cassette from the pBTK622 plasmid via BsaI digestion. Homology flanks were ligated together via BsmBI digestion to create our “rescue" cassette. We confirmed GGA ligation fragment sizes, the 3.7 kb target band consisting of a 1.7 kb *tdk/kan* fragment and 1 kb upstream and downstream flanks, with gel electrophoresis (Figure 5C).

![](https://static.igem.wiki/teams/4342/wiki/tri-gell.png)

**Fig. 5. Synthesis of *ampD* Homology Flanks and *tdk/kan* Transformation Cassette.** **(A)** PCR Amplification of 5’ Upstream Flanks (1 kb) using an annealing temperature gradient of 56°C to 64°C. **(B)** PCR Amplification of 3’ Downstream Flanks (1 kb) using an annealing temperature gradient of 56°C to 64°C. **(C)** PCR Amplification of a BsaI GGA Reaction, ligating *ampD* homology flanks and pBTK622 *tdk/kan* cassette (3.7 kb).

# Test

We added 15 μl of the first GGA reaction to a culture tube containing 500 μl of LB and 35 μl of overnight ADP1 culture. After overnight incubation (30°C, 200 rpm), we plated 100x diluted transformations onto LB-Kanamycin (Kan) plates to confirm the insertion of the *tdk/kan* cassette (Figure 6).

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-6.png)

**Fig. 6. Failed Transformation of the *tdk/kan* Selection Cassette.**  No growth on the +DNA LB-Kan plate shows that the  *tdk/kan*  cassette was not inserted into the  *ampD*  genome location in ADP1. LB plates and -DNA serve as controls for our transformations.

Figure 6 shows that the *tdk/kan* cassette transformation did not work, and repeated attempts continued to fail.

# Learn

We were unable to transform the *tdk/kan* cassette into the *ampD* gene location. Repeated transformations on LB-Kan plates continued to fail. Looking back on our primer design, we found that *ampD* could form an operon with the essential gene *murJ*, and the *tdk/kan* cassette insertion could be cutting off the promoter for *murJ*.

In future primer designs, we checked nearby genes to ensure that knocking out our target gene did not affect ADP1-ISx viability. In Phase 2, we targeted *pbpG* and *acrB*, other genes that contribute to intrinsic β-lactam antibiotic resistance.

Furthermore, we found that using a gradient of annealing temperatures, ranging ± 5°C from the lowest Tm temperature in the set of primers (from NEB Tm calculator), helped improve PCR amplification and purification concentrations. Using a temperature gradient also helped us optimize annealing temperatures for each set of primers, given the intensity of the gel electrophoresis band.

# Phase 2: *pbpG* and *acrB* Engineering

Next, we chose to knock out *pbpG* and *acrB*, ADP1 genes that also contribute to intrinsic β-lactam antibiotic resistance. *pbpG* encodes a penicillin-binding protein involved in peptidoglycan synthesis, and *acrB* encodes proteins involved in efflux pumps [3]. Creating these knockout ADP1 strains demonstrate that these ADP1 genetic engineering protocols are effective. Further data on the [Results Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) show that we successfully made ADP1 more susceptible to Carbenicillin.

# Design

We designed primers to amplify ~1 kb homologous 5’ and 3’ flanks upstream and downstream of *pbpG* and *acrB*. These primers follow the same design as shown in Figure 4. Each set of primers contains a “Golden” primer that attaches BsaI and BsmBI restriction sites to the end of the primer, containing the correct GGA Type Overhang (BsaI) and 4 bp "rescue" scar (BsmBI). These amplified homology flanks, once assembled, are used to modify the ADP1 genome. In the first transformation step, the upstream and downstream flanks are ligated with the *tdk/kan* cassette using GGA via BsaI digestion. In the second transformation step, the upstream and downstream flanks are ligated to each other using GGA via BsmBI digestion.

# Build

We synthesized homology flanks using PCR using our designed *pbpG* and *acrB* primers and ADP1-ISx genomic DNA. We used gel electrophoresis to verify ~1 kb upstream and downstream flank fragment sizes (Figure 7A) and the Zymo DNA Clean and Concentrate Kit to purify PCR products.

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-7.png)

**Fig. 7. Synthesis of  *pbpG*  Homology Flanks and  *tdk/kan*  Transformation Cassettes. (A)** PCR Amplification of  *pbpG*  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 *pbpG* Upstream and Downstream flanks with pBTK622, containing the  *tdk/kan*  cassette.  **Lane 1: 3.7 kb  *tdk/kan*  ligated to homology flanks,**  3.3 kb pBTK622 plasmid, 1.7 kb  *tdk/kan*  cassette, 1 kb homology flanks. Lanes 2 (no T7 DNA Ligase) and 3 (no homology flanks) serve as controls.

Next, we used GGA to ligate the upstream and downstream homology flanks with the *tdk/kan* cassette from the pBTK622 plasmid via BsaI digestion. Homology flanks were ligated together via BsmBI digestion to create our “rescue” cassette. We confirmed GGA ligation fragment sizes, the 3.7 kb target band consisting of a 1.7 kb *tdk/kan* fragment and 1 kb upstream and downstream flanks, with gel electrophoresis (Figure 7B).

# Test

We use the  *tdk/kan*  cassette to select for successful ADP1 transformants. The *kanR* gene allows us to select on kanamycin (Kan), and the *tdk* gene allows us to select on azidothymidine (AZT).

We added 20 μl of the first GGA reaction to a culture tube containing 500 μl of LB and 35 μl of overnight ADP1 culture. After an overnight incubation (30°C, 200 rpm), we plated 100x diluted transformations onto LB-Kan plates to confirm the insertion of the *tdk/kan* cassette (Figure 8).

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-8.png)

**Fig. 8. Successful Transformation of the  *tdk/kan*  Selection Cassette.**  Growth on the +DNA LB-Kan plates shows that the *tdk/kan* cassette was inserted into the  *pbpG*  and  *acrB*  genome location in ADP1. The -DNA LB-Kan plates serve as a negative control for our transformation.

After inoculating a couple of colonies from the +DNA LB-Kan plate, we performed the same transformation process, adding 20 μl of the second GGA reaction to a culture tube containing 500 μl of LB and 35 μl of the overnight modified ADP1 culture, containing the new *tdk/kan* cassette. Next, we plate on LB-AZT instead of LB-Kan, to confirm the deletions of *pbpG* and *acrB*. This transformation inserted our “rescue” cassette, which contained the two synthesized *pbpG* or *acrB* upstream and downstream homology flanks ligated together. Plating on LB-AZT allows us to select for transformants that replaced the *tdk/kan* cassette with the "rescue" cassette since *tdk* makes ADP1 susceptible to AZT.

![](#)

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-9.png)

**Fig. 9. Successful Deletion of *pbpG* and *acrB* from ADP1.**  Growth on the +DNA LB-AZT plates show that the  *pbpG*  and  *acrB*  “rescue” cassettes were inserted into the  *pbpG*  and  *acrB*  genome locations in ADP1. The -DNA LB-AZT plates serve as a negative control for our transformation.

We further confirmed the deletions of *pbpG* and *acrB* from ADP1 via PCR by comparing ADP1 strains to the *tdk/kan* insertion strains and the gene deletion strains (Figure 10).

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-10.png)

**Fig. 10. Confirmation of (A)  *pbpG*  and (B)  *acrB*  Deletion. (A)**  ADP1 shows the endogenous *pbpG* with upstream and downstream flanks (3 kb).  *tdk/kan*  shows the  *tdk/kan*  cassette with upstream and downstream flanks (3.7 kb). ΔpbpG shows the  *pbpG*  deletion, containing only the upstream and downstream flanks (2 kb). The same  *pbpG*  primers were used across all PCRs.  **(B)**  Standard PCR confirmations as shown in (A), with ADP1 showing the endogenous  *acrB*  with upstream and downstream lengths (5.2 kb). The same  *acrB*  primers were used across all PCRs. These PCRs were performed using (A) ADP1 cells or (B) ADP1 genomic DNA as template DNA.

# Learn

We decided to add the entire unpurified GGA reaction (20 μl) instead of saving 5 μl for gel electrophoresis in the transformation step. Confirmation of the GGA reactions via gel electrophoresis was tedious and time-consuming. We believed using the full mixture would help increase transformation frequency.

# Phase 3: *recJ* and ACIAD2049 Engineering

The third phase shows how we knocked out *recJ* and ACIAD2049 in ADP1. *recJ* encodes a single-strand DNA exonuclease that degrades short cytoplasmic DNA [4]. Knocking out *recJ* could improve transformation frequency for smaller donor DNA fragments (< 500 bp) [5]. Knocking out ACIAD2049, a nonessential ADP1 gene, allows us to integrate other DNA sequences of interest into ADP1.

# Design

We designed primers to amplify ~1 kb homologous 5’ and 3’ flanks upstream and downstream of *recJ* and ACIAD2049. These primers follow the same design as shown in Figure 4. Each set of primers contains a “Golden” primer that attaches BsaI and BsmBI restriction sites to the end of the primer, containing the correct GGA Type Overhang (BsaI) and 4 bp "rescue" scar (BsmBI). These amplified homology flanks, once assembled, are used to modify the ADP1 genome. In the first transformation step, the upstream and downstream flanks are ligated with the *tdk/kan* cassette using GGA via BsaI digestion. In the second transformation step, the upstream and downstream flanks are ligated to each other using GGA via BsmBI digestion.

# Build

We synthesized homology flanks using PCR using our designed *recJ* and ACIAD2049 primers and ADP1-ISx genomic DNA. We used gel electrophoresis to verify ~1 kb upstream and downstream flank fragment sizes (Figure 11) and the Zymo DNA Clean and Concentrate Kit to purify PCR products.

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-11.png)

**Fig. 11. Synthesis of  *recJ*  Homology Flanks.**  PCR Amplification of  *recJ*  5’ Upstream Flanks (1 kb) and 3’ Downstream Flanks (1 kb) using an annealing temperature gradient of 53°C to 60°C.

Next, we used GGA to ligate the upstream and downstream homology flanks with the *tdk/kan* cassette from the pBTK622 plasmid via BsaI digestion. We ligated the homology flanks together via BsmBI digestion to create our “rescue” cassette.

# Test

We use the  *tdk/kan*  cassette to select for successful ADP1 transformants. The *kanR* gene allows us to select on kanamycin (Kan), and the *tdk* gene allows us to select on azidothymidine (AZT).

We added 20 μl of the first GGA reaction to a culture tube containing 500 μl of LB and 35 μl of overnight ADP1 culture. After overnight incubation (30°C, 200 rpm), we plated 100x diluted transformations onto LB-Kan plates to confirm the insertion of the *tdk/kan* cassette (Figure 12).

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-12.png)

**Fig. 12. Successful Transformation of the *tdk/kan* Selection Cassette.**  Growth on the +DNA LB-Kan plate shows that the  *tdk/kan*  cassette was inserted into the  *recJ*  genome location in ADP1. The -DNA LB-Kan plate serves as a negative control for our transformation.

After inoculating a couple of colonies from the +DNA LB-Kan plate, we performed the same transformation process, adding 20 μl of the second GGA reaction to a culture tube containing 500 μl of LB and 35 μl of the overnight modified ADP1 culture, containing the new *tdk/kan* cassette. Next, we plate on LB-AZT instead of LB-Kan, to confirm the deletions of *recJ* (Figure 13) and ACIAD2049. This transformation inserted our “rescue” cassette, which contained the synthesized *recJ* upstream and downstream homology flanks ligated together. Plating on LB-AZT allows us to select for transformants that replaced the *tdk/kan* cassette with the "rescue" cassette since *tdk* makes ADP1 susceptible to AZT.

![](https://static.igem.wiki/teams/4342/wiki/eng-fig-13.png)

**Fig. 13. Successful Deletion of *recJ* from ADP1.**  Growth on the +DNA LB-AZT plate shows that the  *recJ*  “rescue” cassette was inserted into the  *recJ*  genome location in ADP1. The -DNA LB-AZT plate serves as a negative control for our transformation.

# Learn

Using this two-step ADP1 genetic engineering protocol, we were able to reliably modify ADP1’s genome. Selecting on LB-Kan and LB-AZT plates demonstrates how we use the *tdk/kan* cassette to confirm our transformations and genome modifications. We learned that we could easily design different “rescue” DNA sequences to replace the *tdk/kan* cassette, inserted in the place of each endogenous ADP1 gene. **We followed these engineering phases to construct our ADP1 biosensors for detecting Antibiotic Resistance Genes and White Nose Syndrome.**

# References

[1] Suárez, G. A., Dugan, K. R., Renda, B. A., Leonard, S. P., Gangavarapu, L. S., & Barrick, J. E. (2020). Rapid and assured genetic engineering methods applied to *Acinetobacter baylyi* ADP1 genome streamlining. *Nucleic acids research*, 48(8), 4585-4600. https://doi.org/10.1093/nar/gkaa204

[2] Biggs, B. W., Bedore, S. R., Arvay, E., Huang, S., Subramanian, H., McIntyre, E. A., ... & Tyo, K. E. J. (2020). Development of a genetic toolset for the highly engineerable and metabolically versatile *Acinetobacter baylyi* ADP1. *Nucleic acids research*, 48(9), 5169-5182. https://doi.org/10.1093/nar/gkaa167

[3] 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

[4] Han, E. S., Cooper, D. L., Persky, N. S., Sutera Jr, V. A., Whitaker, R. D., Montello, M. L., & Lovett, S. T. (2006). *RecJ* exonuclease: substrates, products and interaction with SSB. *Nucleic acids research*, 34(4), 1084-1091. https://doi.org/10.1093/nar/gkj503

[5] Overballe-Petersen, S., Harms, K., Orlando, L. A., Mayar, J. V. M., Rasmussen, S., Dahl, T. W., ... & Willerslev, E. (2013). Bacterial natural transformation by highly fragmented and damaged DNA. *Proceedings of the National Academy of Sciences*, 110(49), 19860-19865. https://doi.org/10.1073/pnas.1315278110

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# Experiments

# Procedures & Protocols

# Purpose

This year, the UT Austin iGEM team worked with *Acinetobacter baylyi* ADP1 as our primary chassis organism.Handling ADP1 in the lab requires a **unique** set of protocols that are designed to take advantage of its **natural competence**. These unique protocols can be used alongside standard molecular biology procedures including PCR, Golden Gate Assembly, and plating techniques, to **efficiently engineer ADP1**. The experiments below describe both ADP1-specific protocols and standard molecular biology protocols needed to work with ADP1. We hope that providing the iGEM community with these procedures will promote the usage of ADP1 in future iGEM projects. Through our iGEM project, we show that **ADP1 is a viable and advantageous chassis organism for synthetic biology and can be used by future iGEM teams for their own creative and scientific endeavors.**

# Golden Gate Assembly (NEB)

This procedure allows you to ligate multiple DNA sequences together to create integration or rescue cassettes, which are used to engineer the ADP1 genome.

**BsaI Digestion**

1. Pipette the following into a PCR tube:

* 2 μL of 10x T4 DNA ligase buffer (NEB: M0202S)
* 1 μL of BsaI-HF (NEB: R3535S)
* 1 μL of T7 DNA ligase (NEB: M0318S)
* 250 ng plasmid (pBTK622)
* 150 ng 5' flank homology
* 150 ng 3' flank homology
* ddH2O to 20 μL rxn total volume

2. Place the tube into a thermocycler and run using the below settings:
   1. 37°C, 5:00
   2. 16°C, 5:00
   3. Goto 1, 30x
   4. 55°C, 5:00
   5. 80°C, 5:00
   6. 12°C, ∞

‍

**BsmBI Digestion**

1. Pipette the following into a PCR tube:

* 2 μL of 10x T4 DNA ligase buffer (NEB: M0202S)
* 1 μL of BsmBI
* 1 μL of T7 DNA ligase (NEB: M0318S)
* 100 ng 5' flank homology
* 100 ng 3' flank homology
* ddH2O to 20 μL rxn total volume

2. Place the tube into a thermocycler and run using the same settings used for BsaI digestion, changing the first step to 42°C

‍

# Creating the Exogenous DNA for ADP1 Transformation

These procedures allow you to genetically engineer the ADP1 chromosome. The first procedure creates the **integration cassette** and the latter two procedures create the **rescue cassettes** needed for ADP1 transformation.

***tdk/kan*  Cassette Insertion**

1. In a PCR tube, set up the BsaI Golden Gate Ligation Reaction with the following:

* 2 μL of 10X T4 DNA ligase buffer (NEB: M0202S)
* 1 μL BsaI-HF (NEB: R3535S)
* 1 μL of T7 DNA ligase (NEB: M0318S)
* 250 ng pBTK622 plasmid
* 150 ng 5’flank homology (for ~1000bp)
* 150 ng 3’flank homology (for ~1000bp)
* ddH2O to 20 µL rxn total volume

2. Run thermocycler with the following settings:

* 37°C, 5:00
* 16°C, 5:00
* Goto 1, 30X
* 55°C, 5:00
* 80°C, 5:00
* 12°C, until the reaction timer ends

***tdk/kan*  Cassette "Rescue" Deletion**

1. In a PCR tube, set up the BsmBI Golden Gate Assembly with the following:

* 2 μL of 10X T4 DNA ligase buffer (NEB: M0202S)
* 1 μL BsmBI (NEB: R0580S)
* 1 μL of T7 DNA ligase (NEB: M0318S)
* 100 ng 5’flank homology
* 100 ng 3’flank homology
* ddH2O to 20 µL rxn total volume

2. Run thermocycler with the following settings:

* 42°C, 5:00
* 16°C, 5:00
* Goto 1, 30X
* 55°C, 5:00
* 80°C, 5:00
* 12°C, until the reaction timer ends

***tdk/kan*  Cassette "Rescue" Deletion with Genetic Device**

1. In a PCR tube, set up the BsmBI Golden Gate Assembly with the following:

* 2 μL of 10X T4 DNA ligase buffer (NEB: M0202S)
* 1 μL BsaI-HF (NEB: R0580S)
* 1 μL of T7 DNA ligase (NEB: M0318S)
* 100 ng 5’flank homology
* 100 ng 3’flank homology
* x ng insert DNA of genetic device
* ddH2O to 20 µL rxn total volume

2. Run thermocycler with the following settings:

* 42°C, 5:00
* 16°C, 5:00
* Goto 1, 30X
* 55°C, 5:00
* 80°C, 5:00
* 12°C, until the reaction timer ends

# General ADP1 Transformation and Genome Manipulation

This procedure is used to engineer the ADP1 chromosome using exogenous DNA.

1. Grow an overnight culture of the ADP1 strain you are manipulating.
2. Prepare two culture tubes with 500 μL LB media + 35 μL of overnight culture. Label one tube “- DNA” and the other “+ DNA”.
3. Add 50-100 ng of the transformant DNA to the tube labeled “+ DNA.”
4. Incubate the tubes at 30°C and shake at 200 rpm overnight.
5. Dilute the “+ DNA” mixture 100-fold in sterile saline and plate 50 μL onto selective media. Repeat for the "- DNA" mixture.
6. Incubate plates overnight at 30°C.
7. Select 2-3 colonies from the “+ DNA” plate, and inoculate an overnight culture. Incubate at 30°C and 200 rpm.
8. Using the overnight culture, make glycerol stocks of each biological replicate.

‍

# Overnight Culture

1. Obtain a standard culture tube for each strain to be inoculated, and an additional tube for the negative control.
2. Label one tube “- Control” and the other tubes according to the strain that they will contain<./li>- Add 5 mL of LB to each of the tubes.
   - If the growth medium requires an antibiotic, add 5 μL, (1000x of the stock concentration), of desired antibiotic to the culture tube.
   - Pipette 2 μL of a glycerol stock into each tube except for the - Control tube. Vortex briefly. If inoculating from a plate, select a single colony using a sterile loop and swirl the loop in the media.
   - Place the tubes in either a 30°C (ADP1) or 37°C (*E. coli* ) incubators at 250 rpm and shake overnight.

# Glycerol Stocks

1. Grow an overnight culture of the strain you wish to freeze.
2. Obtain and label cryovials with the name of the strain that will be frozen.
3. Working around a lit Bunsen burner, add 200 μL of 80% Glycerol to the cryovial. Glycerol is thick, so use a P1000 pipette. Pull and push the pipette trigger slowly when extracting and dispensing the liquid.
4. Using an P1000 pipette, add 1 mL of overnight culture to the labeled cryovial.
5. Close the cryovial and vortex vigorously for ~10 seconds.
6. Place the cryovials in a -80°C freezer.

# Polymerase Chain Reaction (PCR)

1. For a 50 μL reaction, pipette the following reagents into a 1.7 mL microcentrifuge tube to create a master mix:

* 2.5 μL of 10 μM upstream forward primer
* 2.5 μL of 10 μM downstream reverse primer
* 10 μL of 5x Phusion buffer
* 1 μL of 10 mM dNTPs
* 1.5 μL of Phusion Buffer
* 0.5 μL of Phusion Polymerase
* 32 μL of ddH2O
* NOTE: The above quantities can be multiplied if more than one PCR needs to be performed. For example, a master mix for 3 PCRs would include 3 times the amount of each reagent.

2. Pipette 49 μL of master mix into a PCR tube.
3. Pipette 1 μL of template DNA into the PCR tube. For control tubes, pipette 1 μL of ddH2O instead.
4. Place the PCR tube in the thermocycler and run the following conditions

1. Denaturation: 98°C, 0:10
2. Annealing: Tm (melting temperature) of Primers, 0:30
3. Extension: 72˚C, 0:30
4. Goto 1, 34x
5. 72˚C, 5:00
6. 12˚C, ∞

# Gel Electrophoresis

**Making the Gel**

‍

1. Add 50 mL of TAE buffer to a 250 mL Erlenmeyer flask.
2. Weigh out .5 grams of Agarose (1% Agarose Gel) and pour it into the Erlenmeyer flask.
3. Microwave the mixture for 1:30.
4. Add 2.5 μL (0.005%) of Invitrogen’s SYBR Safe DNA Gel Stain to the heated mixture.
5. Swirl the mixture to disperse the dye.
6. Pour the mixture into an agarose gel mold containing the desired number of wells.
7. Wait ~20 minutes for the mixture to solidify.
8. Once the gel has solidified, place the gel into the gel rig and pour TAE buffer until the gel is submerged.
9. Proceed to “Loading the gel.”

‍

**Loading the Gel**

‍

1. Add 6 μl of 1 kb+ ladder to the leftmost well.
2. Add 1 μl of NEB purple loading dye to 5 μl of PCR product and pipette up and down to homogenize the mixture.
3. Repeat step 2 for all samples.
4. Once all samples have been properly prepared, load each sample into the gel starting from left to right, noting which sample goes into each lane.
5. After each sample has been loaded, proceed to “Running the Gel.”

‍

**Running the Gel**

‍

1. Once all samples have been loaded, attach the lid to the gel rig and connect it to a power supply.
2. Set the power to 120 V and let the gel run for 20-30 minutes. (Note: 25-30 minutes is ideal for products longer than 2 kb because it allows for greater separation of the ladder)
3. After 20-30 minutes, pause or turn off the power supply, unload the gel tray, and place the gel into a transilluminator.
4. Proceed to "Analyzing the Gel."

‍

**Analyzing the Gel**

‍

1. Place the tray in a transilluminator and run a protocol that is compatible with Invitrogen's SYBR Safe dye.
2. Save the image of the gel.

‍

# Competent Cells

1. Remove one tube of cells from the freezer for each transformation reaction, plus appropriate controls.
2. Thaw tubes on ice.
3. Add transforming DNA (up to 25 ng, or 0.5-2 μL of a miniprepped plasmid) and incubate on ice for 30 minutes.

* For transforming cloning reactions (e.g. from Gibson Assembly), it is possible to add up to 5 μL of reaction directly to the cells without any purification steps.

4. Transfer tubes to a 42°C heat block or water bath for  **EXACTLY**  90 seconds.

* If using a heat block, make sure to fill the wells with diH2O to ensure efficient heat transfer.

5. Immediately transfer tubes to ice, cool 1-2 minutes.
6. Add 450 μL SOC medium to each tube, and shake at 200-250 RPM for 1 hour.

* For transformations in Eppendorf tubes, place the tubes inside an empty 250 mL flask, and incubate in the large shaking incubators.

7. Plate at least two different volumes (10 μL and 50 μL) of each reaction on appropriate antibiotic plates.

* If your efficiencies tend to be on the low side, pellet your cells by centrifuging for 5 minutes at 3000 rpm. Resuspend in 100 μL of saline, and plate the entire mixture.

# Spot Plating

This procedure is used to determine the transformation efficiency and detction threshold of ADP1 .

1. Fill each of the wells of the 96-well plate that you will be using with 180 µL Saline/PBS (see image on right).
2. Fill the top row of your plate with 200 µL sample.
3. Using the P200 pipet, perform serial dilutions of 20 µL into each well (20 in 200 = 1:10 dilutions) -  **Change tips between each transfer!**
4. Using your P20 pipette - pipette up to 5 µL of sample, line it up with the grid on your plate, touch the pipette tips to the plate, and slowly depress the plunger to deposit a row of droplets onto the agar.

* If your sample spreads into another sample, your plate is not dry enough! Stop what you are doing and let the plates dry.

5. Plate all your samples in triplicate from the same set of dilutions.
6. Allow your spots to dry, and transfer them to the appropriate incubator to grow.

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© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)
The repository used to create this website is available at [gitlab.igem.org/2022/austin-utexas.](https://gitlab.igem.org/2022/austin-utexas)

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# Results

# Results

[• Foundational Advance](#foundational-advance)
[• Antibiotic Resistance Detectors](#Anitbiotic-Resistance-Detectors)
[• White Nose Syndrome Detector](#White-Nose-Syndrome)
[• Conclusion](#Conc)

# Foundational Advance

# Introduction

To improve ADP1’s viability as a chassis organism, we knocked out  *pbpG*  and  *acrB*, two intrinsic genes that contribute to intrinsic β-lactam antibiotic resistance. *pbpG* encodes a penicillin-binding protein involved in peptidoglycan synthesis, and *acrB* encodes proteins involved in efflux pumps [1]. After deleting these genes using our ADP1 Genetic Engineering protocol, we designed experiments to test whether ADP1 became more sensitive to β-lactam antibiotics, such as Carbenicillin and Ampicillin. We measured the minimum inhibitory concentration (MIC) of each antibiotic for our engineered ADP1 strains. Our results indicate that several different “Wild-Type” ADP1 strains are already sensitive to Carbenicillin and Ampicillin, but knocking out *pbpG* and *acrB* from these strains increased ADP1’s susceptibility to these antibiotics.

# Minimum Inhibitory Concentration (MIC) Experiments

![](https://static.igem.wiki/teams/4342/wiki/results/mic.png)

**Fig. 1. *ΔpbpG* and *ΔacrB* Carbenicillin MIC Growth Assay.**  Plates were streaked out overnight. *ΔpbpG* and *ΔacrB* biological triplicates, ADP1-ISx biological duplicates, and one *E. coli* MG1655 colony were scraped and diluted in 1 mL saline. 2 μl of the diluted sample was inoculated into standard culture tubes containing 5 mL of LB and 2-fold concentrations of Carbenicillin, ranging from 2-128 μg/mL . Cultures were grown at 30°C for 24 hours. *E. coli* was used as a negative control.

**Δ*pbpG* vs. Δ*acrB* Carbenicillin MIC**

Figure 1 shows both *ΔpbpG* and *ΔacrB* have a Carbenicillin MIC of ~2 μg/mL, the lowest concentration at which ADP1 shows noticeable growth. An interesting finding was that ADP1-ISx has an MIC of 16 μg/mL since we expected ADP1-ISx to be intinsically resistant to Carbenicillin. Although deleting *ΔpbpG* and *ΔacrB* did make ADP1 more sensitive to Carbenicillin, these results made us think about whether this domesticated ADP1-ISx strain [2] had removed some sort of DNA sequence conferring Carbenicillin resistance. Next, we tested ADP1-ISx vs. ADP1-ISx's ancestral strain, named "ADP1-WT" for the purpose of these experiments.

![](https://static.igem.wiki/teams/4342/wiki/results/adp1-v-wt.png)

**Fig. 2. ADP1-WT and ADP1-ISx Carbenicillin MIC Growth Assay.** Plates were streaked out overnight. ADP1-WT and ADP1-ISx biological triplicates, one *ΔpbpG* colony, and one *E. coli* MG1655 colony were scraped and diluted in 1 mL saline. 2 μl of the diluted sample was inoculated into standard culture tubes containing 5 mL of LB and 2-fold concentrations of Carbenicillin, ranging from 2-128 μg/mL . Cultures were grown at 30°C for 24 hours. *E. coli* was used as a negative control.

**ADP1-WT vs. ADP1-ISx Carbenicillin MIC**

Figure 2 shows both ADP1-WT and ADP1-ISx have a Carbenicillin MIC of ~8 μg/mL, showing no noticeable differences in MIC between the two ADP1 strains. As our results continued to show that ADP1-WT and ADP1-ISx were susceptible to Carbenicillin, we took a closer look at Gomez and Neyfakh's work [1] to try to replicate their experiment as closely as possible. We obtained a culture of BD413, which was the "Wild-Type" ADP1 strain Gomez and Neyfakh used to demonstrate ADP1's intrinsic resistance to β-lactam antibiotics. Another modification was inoculating fresh cells grown in LB overnight into LB cultures containing antibiotics, rather than directly inoculating diluted colony mixtures. Additionally, we decided to start using Ampicillin, rather than Carbenicillin.

![](https://static.igem.wiki/teams/4342/wiki/results/mic-3.png)

**Fig. 3. Ampcillin MIC Pilot Growth Assay.** Plates were streaked out overnight. Individual colonies for each strain were scraped and diluted in 1 mL saline. 5 μl of the diluted sample was inoculated into standard culture tubes containing 5 mL of LB. Fresh cells were diluted 1000-fold into standard culture tubes containing LB and 2-fold concentrations of Ampicillin, ranging from 2-128 μg/mL. Cultures were grown at 30°C for 24 hours. *E. coli* was used as a negative control.

**Ampicillin MIC Pilot**

As a preliminary experiment, we tested a variety of ADP1 strains to test their resistance to Ampicillin. Figure 3 shows both the ADP1-WT and ADP1-ISx strains exhibited an MIC of about 2~4 μg/mL, but our knockout *pbpG* and *acrB* strains exhibited an increased susceptibility to Ampicillin, with an MIC < 2 μg/mL. Using the same Ampicillin stocks for the final MIC experiment, we included a positive control, pUC19 (carrying an *ampR* gene) expressed in *E. coli*, to confirm our Ampicillin concentrations were correct. Our last thought was to test whether growth temperature had an impact on ADP1 antibiotic resistance.

![](https://static.igem.wiki/teams/4342/wiki/results/mic-4.png)

**Fig. 4. BD413 30°C vs. 37°C Ampicillin MIC Growth Assay.** Plates were streaked out overnight. Single BD413 and *E. coli* MG1655 colonies were scraped and diluted in 1 mL saline. 5 μl of the diluted sample was inoculated into standard culture tubes containing 5 mL of LB. Fresh cells were diluted 1000-fold into standard culture tubes containing LB and 2-fold concentrations of Ampicillin, ranging from 2-128 μg/mL. Cultures were grown at 30°C for 24 hours. *E. coli* was used as a negative control.

**BD413 30°C vs. 37°C Ampicillin MIC**

Figure 4 shows that BD413 had an MIC ~2 μg/mL in both 30°C and 37°C. Growth in our positive control confirmed correct Ampicillin concentrations.

Our data consistently shows that "Wild-Type" ADP1 strains, including ADP1-ISx, ADP1-WT, and BD413 do not have intrinsic resistance to β-lactam antibiotics, but future experiments involving replicates for each strain are necessary to confirm our findings. However, our findings show that knocking out genes that contribute to β-lactam antibiotic resistance can increase ADP1's susceptibility to β-lactams (Figure 1), providing an additional selection method for ADP1.

# Antibiotic Resistance Detectors

Before developing the *Pseudogymnoscus destructans* detector, it was important to first prove that ADP1 could be engineered to detect environmental DNA. To accomplish this, we created a novel biosensor for the detection of the *TEM-1* antibiotic resistance gene and adapted the work done by De Vries et al. [3] to create a biosensor for the detection of the *nptII* antibiotic resistance gene. De Vries et al. [3] created a system to detect the *nptII* (Kanamycin resistance) gene by inserting a plasmid into ADP1 that contained a mutated, non-functional version of the gene. We adapted this system by integrating the nonfunctional gene into ADP1's genome as opposed to putting it on a plasmid. In this section, we will reiterate the results found on our [Proof of Concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D) page and discuss additional experiments we performed to better characterize ADP1’s ability to detect environmental DNA.

# Engineering Confirmation

Both the *TEM-1* and the *nptII* biosensor were constructed by inserting a broken version of the target gene into ADP1’s genome. For an in-depth explanation of how these detectors were developed, see our [Proof of Concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D) Page. We confirmed that both biosensors were engineered properly by amplifying the *nptII* and *TEM-1* broken genes from purified genomic DNA of each biosensor. Figures 5a and 5b show that the ADP1 genomes of both biosensors contained the correct length of the non-functional gene, confirming that we properly engineered the detector. Nanopore sequencing provided additional confirmation that both detectors were successfully engineered.

![](https://static.igem.wiki/teams/4342/wiki/poc-fig-3.png)

**Fig. 5. Confirmation of (A) *TEM-1* and (B) *nptII* Detectors. (A)** *TEM-1* Detector PCR contains the *TEM-1* Broken Gene with the expected length of 1133 bp. **(B)** *nptII* Detector PCR contains the *nptII* Broken Gene with the expected length of 1639 bp.

# Detecting the Target Gene

After confirming proper engineering, a growth assay was performed on each biosensor. The goal of this assay was to determine if the biosensors could detect the WT target gene while simultaneously confirming that the broken gene in the chromosome was nonfunctional.

For this growth assay, the detector strains were grown in three separate solutions: one with the target gene (+DNA), one with no DNA (-DNA), and one with an off-target gene (*nptII* for the *TEM-1* detector and *TEM-1* for the *nptII* detector). After an overnight incubation period, each culture was plated onto non-selective plates LB plates and selective LB-Amp or LB-Kan plates (Ampicillin for the *TEM-1* detector and Kanamycin for the *nptII*  detector). Figures 6 and 7 show that the *TEM-1* and *nptII* biosensors were unable to grow on the selective plate when no DNA (-DNA) was added, proving that the mutated version of the *TEM-1* and *nptII* genes inserted into ADP1’s chromosome were non-functional. Additionally, the plates highlighted in orange show that each strain grew successfully on the selective plate when the target gene was added, confirming that the ADP1 cells detected the WT target gene.

# *TEM-1* Detector

LB

Off-target DNA

*TEM-1* Gene

-DNA

LB+Amp

![](https://static.igem.wiki/teams/4342/wiki/tem-1-plates.png)

**Fig. 6. Detection of the  *TEM-1*  Gene.** Each column shows plates of the the *TEM-1* detector strain grown in the presence of the following DNA sequences. From left to right: the target *TEM-1* gene, no DNA, and off-target DNA (*nptII* gene PCR product from pKD13). The top row shows the biosensor's ability to grow on Ampicillin. The bottom row shows the biosensor's ability to grow on LB. The highlighted plate shows the expected result; The *TEM-1* detector strain was able to grow on ampicillin when the WT target gene was added.

# *NptII* Detector

LB+Kan

LB

*NptII* Gene

-DNA

Off-target DNA

![](https://static.igem.wiki/teams/4342/wiki/nptii-plates.png)

**Fig. 7. Target Gene Detection.** Each column shows plates of the the *nptII* detector strain grown in the presence of the following DNA sequences. From left to right: the target *nptII*gene, no DNA (-DNA), and off-target DNA (*TEM-1* PCR product from pBTK404). The top row of plates shows the biosensor's ability to grow on Kanamycin plates. The bottom row shows the biosensor's ability to grow on LB. The highlighted plate shows the expected result; The *nptII* detector strain was able to grow on Kanamycin when the target gene was added.

It is important to note that whenever the off-target gene was added to the *nptII* detector strain, ADP1 was unable to grow on selective plates, but when the off-target gene was added to the *TEM-1* detector, ADP1 shows some growth on the selective plate. In Figure 7, the top right plate shows this result. The off-target DNA we used to test the *TEM-1* detector was PCR product of the *nptII* gene. The template DNA for this PCR was the pKD13 plasmid, which happens to have the *TEM-1* gene as its backbone. We believe that growth on the yellow highlighted plate could have occurred because our PCR cleanup failed to remove all of the template DNA. One could do DpnI digestion to completely remove all the template DNA.

# System Characterization

To better characterize ADP1’s ability to detect environmental DNA, we tested the biosensor against target DNA concentrations (ng/mL) ranging from 0.0001 ng/mL to 100 ng/mL. Figure 8 shows that both detectors could detect the target gene at 0.001 ng/mL. Additional tests showed that both biosensors could not detect the target gene at .0001 ng/mL, suggesting our system's detection threshold to be ~0.001 ng/mL.

![](https://static.igem.wiki/teams/4342/wiki/results/results/results/detection-threshold-pics.png)

**Fig. 8. Detection Threshold Determination.**  *TEM-1*  Detector (left),  *nptII*  detector (right).

We also calculated the transformation efficiencies for each concentration by dividing the selective CFU counts by the amount of DNA in the solution. Figure 9 graphs the transformation frequency as a function of the DNA concentration. The graphs for both detectors show that as the concentration increases ten-fold, transformation frequency also increases ten-fold. This relationship, however, seems to level off near the 10 and 100 ng/mL concentrations, suggesting that our detectors possess a maximum transformation frequency.

![](https://static.igem.wiki/teams/4342/wiki/graphs.png)

**Fig. 9. Transformation Frequency Graphs. (A)** *TEM-1*  Transformation Frequency and **(B)**  *nptII*  Transformation Frequency.

# Detecting DNA in Mixed DNA Samples

![](https://static.igem.wiki/teams/4342/wiki/results-graphs-2.png)

**Fig. 10. Transformation Frequency in the Presence of Mixed DNA.** **(A)** *nptII* Detector and **(B)** *TEM-1* Detector parsing through 10-fold amounts of *E. coli* genomic DNA relative to WT Gene PCR Product. All samples had ~1 ng/mL of the WT Gene added to the transformation mixture.

As we continued to further characterize our antibiotic resistance detectors, one of our goals was to determine whether ADP1 could parse through mixtures of random DNA to detect the correct WT gene. Figure 10 shows that there is not a significant difference in Transformation Frequency, when adding up to 1000-fold more *E. coli* genomic DNA to the transformation culture. This shows how ADP1 has potential to function efficiently in more realistic environments to detect DNA sequences of interest. Future experiments could test >1000-fold amounts of random DNA, multiple types of random DNA, and DNA detection thresholds in co-cultures.

To summarize, the *TEM-1* and *nptII* biosensors we developed were confirmed to have been engineered properly and proved that they could successfully detect the target gene in a lab setting. Furthermore, we determined that both biosensors were successful, provided that the WT target gene was present at a concentration of at least .001 ng/mL. The ADP1 transformation frequency data indicates that the calculated transformation frequency is proportional to the concentration of the target gene in the environment. Finally, ADP1 transformation frequency not being affected by random DNA demonstrates how ADP1 can efficiently detect the correct WT target gene in the presence of other DNA.

# White Nose Syndrome Detector

We designed a modular detection construct that utilizes a YFP gene and repressor system in conjunction with a *tdk/kan* cassette to detect environmental DNA from *Pseudogymnoascus destructans*. Figure 11 shows the *tdk/kan* cassette and *cymR* repressor between the *P. destructans* homology regions. Additionally, the *cymR* associated YFP gene is inserted into a completely seperate part of the chromosome. This YFP gene is repressed by the *cymR* repressor until the target *P. destructans* DNA sequence is identified in the environment. At this point, ADP1 homologous recombination occurs, removing the *tdk/kan* cassette and the *cymR* repressor gene. ADP1 cells that successfully knocked out the *tdk/kan* cassette can then selectively grow on AZT and produce a YFP signal.

![](https://static.igem.wiki/teams/4342/wiki/wns-detector-construct.png)

**Fig. 11. *P. destructans* Modular Construct Designed to Detect White Nose Syndrome.**

First, we identified a YFP and repressor system that was compatible with ADP1. We tested the *cymR*, *lacI*, *vanR*, and *betI* YFP repressor systems designed by Meyer *et al.* [4]. The YFP genes for these systems are controlled by promoters specific to each repressor. For example, the *cymR*-compatible YFP gene is controlled by a *cymR*-specific promoter. We attempted to individually integrate all four YFP variatons into ADP1's chromosome. This was completed by replacing the ADP1 *acrB* gene with individual YFP genes. Figure 10 shows that the *CymR*, *LacI* and *BetI* YFP gene insertions integrated successfully, and the *VanR* insertion did not integrate successfully.

![](https://static.igem.wiki/teams/4342/wiki/yfp-insertion-corrected.png)

**Fig. 12. YFP Insertions.**  From left to right: Ladder, Control, ADP1-ISx, *cymR-YFP*, *vanR-YFP*, *lacI-YFP*, and *betI-YFP*. Expected length for each YFP variation was ~3000 base pairs.

To determine which YFP variations were functional, the fluorescence intensity of each engineered YFP strain was measured with a plate reader using an excitation wavelength of 488 nm and emisssion wavelength of 530 nm. The *YFP* fluorescence measurements were compared to the ADP1-ISx strain. As seen in Figure 11, the  *cymR*  and  *lacI*  YFP genes had fluorescence readings greater than ISx’s basal fluorescence, signaling that these YFP genes were compatible in ADP1-ISx. This data led to our decision to use the  *cymR-YFP* repressor system for the White Nose Syndrome detector construct.

![](https://static.igem.wiki/teams/4342/wiki/parts/parts/yfp-fluorescence-data.png)

**Fig. 13. YFP Average Fluorescence.**

After identifying the  *cymR-YFP*  repressor system, we began create the more complex White Nose Syndrome detector construct. To create this detector, we ran three different BsaI Golden Gate Assembly (GGA) reactions. Figure 13 shows how we used these three reactions to create the detector construct.

![](https://static.igem.wiki/teams/4342/wiki/results/pd-detector.png)

**Fig. 14. Creating the  *P. destructans* Detector Construct.** Reaction 1 ligates the ACIAD2049 Upstream part with the  *P. destructans*  Upstream part. Reaction 2 ligates the  *cymR*  repressor, the  *P. destructans*  Downstream part and the ACIAD2049 Downstream part together. Reaction 3 ligates the Reaction 1 and Reaction 2 products with the *tdk/kan* cassette, creating the entire detector construct. All reactions used BsaI digestion.

After running the three GGA reactions, we succesfully produced the entire detector construct. Figure 15 shows a bright band ~7 kb, the expected size of Reaction 3. The intensity of this band indicates that the primary DNA sequence amplified in Reaction 3 is the correct White Nose Syndrome detector construct. This Golden Gate product was then transformed into ADP1's genome using Step One of the ADP1 Genetic Engineering protocol outlined on our [Parts Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D).

![](https://static.igem.wiki/teams/4342/wiki/results/results/white-background.png)

**Fig. 15. Confirmation of  *P. destructans*  Detector Assembly.** From left to right: Ladder, Reaction 3 GGA Product. Expected length of the White Nose Syndrome Detector is ~7 kb.

To integrate the *P. destructans* detector cassette into ADP1's genome, we added 15 μl of the above GGA reaction to a standard culture tube containing 500 μl of LB and 35 μl of an overnight culture of ADP1 cells with the *cymR*-controlled  *YFP*  gene. After overnight incubation (30°C, 200 rpm), we plated 100x diluted transformations onto LB-Kanamycin (Kan) plates to select for cells that integrated the construct. The plates in Figure 16 demonstrate that some cells successfully integrated the detection cassette.

![](https://static.igem.wiki/teams/4342/wiki/results/results/pd-detector-plates.png)

**Fig. 16. Inserting the White Nose Syndrome Detector Cassette into ADP1's Genome.**

We picked 3 colonies from the highlighted LB-Kan plate to inoculate and make glycerol stocks. We attempted to test these WNS detector strains and their ability to detect the target *P. destructans* gene. However, all of the tests we ran proved to be inconclusive. Our results suggest the detector strains may not have been properly engineered. To test this, we ran a growth assay to determine if any of the *P. destructans* triplicates (034A, 034B, 034C) produced the expected growth pattern (Figure 17). We streaked ADP1-ISx, ADP1 containing an Integration *tdk/kan* cassette and the three *P. destructans* detector strains (034A, 034B, and 034C) onto both Kanamycin and AZT plates. A properly engineered WNS detector strain would show colony growth on the LB-Kan plates while showing no growth on the LB-AZT plates. The 034A and 034C strains did not show this growth pattern, but 034B proved to be promising. This strain was susceptible to AZT, and the zoomed in image on Figure 17 shows very small colonies, suggesting that the 034B strain may possess the correct White Nose Syndrome detector construct. We are planning to sequence the entire *P. destructans* Detector 034B strain to determine if the proper construct was integrated into the ADP1 genome. We hope to have these results by the Grand Jamboree.

![](https://static.igem.wiki/teams/4342/wiki/results/streak-figure.png)

**Fig. 17. Streaks of Detector Strains on Kanamycin and AZT**

# Conclusion

This year, the 2022 UT Austin iGEM team improved ADP1 as a chassis organism for synthetic biology by successfully deleting the *acrB*, *pbpG*, ACIAD2049, and *recJ* genes. Additionally, we characterized ADP1’s susceptibility to β-lactam antibiotics and documented how deleting the *pbpG* and *acrB* genes increase β-lactam susceptibility. We also demonstrated how to engineer a ADP1-based biosensor by creating two antibiotic resistance biosensors. These biosensors were capable of detecting exogenous DNA sequences in a lab setting. Furthermore, we show that ADP1 can efficiently detect the target DNA sequence in the presence of other DNA sequences. Finally, we designed and successfully created the DNA sequence required to make the White Nose Syndrome Detector, but we were unable to confirm whether we successfully inserted the complex construct into ADP1’s chromosome. Future directions for this project include confirming the successful creation of the White Nose Syndrome Biosensor, testing our biosensors' abilties to detect both synthetic and environmental *P. destructans* DNA in realistic environments, and further characterizing ADP1's natural competence capabilities.

# 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] Suárez, G. A., Dugan, K. R., Renda, B. A., Leonard, S. P., Gangavarapu, L. S., & Barrick, J. E. (2020). Rapid and assured genetic engineering methods applied to *Acinetobacter baylyi* ADP1 genome streamlining. *Nucleic acids research*, 48(8), 4585-4600. https://doi.org/10.1093/nar/gkaa204

[3] 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

[4] 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

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