iGEM 2023 — Team ucsc

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![Education graphic.](https://static.igem.wiki/teams/4592/wiki/education/bookcat.png)

## Notebook

---

### **June**

## Week of 6/26/2023

1. **Fundraising/Planning Team**

1. Researching arrangements for travel to iGEM Jamboree
2. Filling out funding applications

2. **Wiki Team**

1. Initial writing of project description, attributions, notebooks, safety page, and human practices
2. Research on iGEM criteria
3. Developed a basic understanding of HTML and CSS to be able to design and edit the wiki moving forward
4. Wiki edits

1. header, footer template
2. notebook page and project description page skeletons

5. Social media: started project reveal

3. **Dry Lab Team**

1. Developing a comprehensive range of project approaches
2. Collecting protocols: culturing *M. Aeruginosa* in optimized conditions for toxin production
3. Met with CA Department of Water Resources for project development and possible future approval/viability for deployment
4. Guidelines for the genetic engineering of *Microcystis* communities based on phage and conjugative plasmids

1. methods for the evaluation of success metrics (toxin concentrations, cell viability, propagation rates)

5. Drafted various protocols related to molecular cloning, phage assembly, various assays including HPLC protocol for studying degradation products
6. Coordinated meeting with Dr. Glenn Milhauser to discuss HPLC methods, usage, protocols, etc.

4. **Wet Lab Team**

1. Made chemicompetent *E. coli* cells to be used the rest of the summer
2. Transformed chemicompetent *E. coli* to check competency
3. Organized the lab space
4. Replenish commonly used reagents
5. Prepped, cleaned, and performed maintenance on the autoclave
6. Took inventory of lab reagents and materials

### **July**

## Week of 7/3/2023

1. **Wiki Team**

1. Edited existing notebook branch to isolate notebook updates from main
2. Started team branch to keep updates to team page and associated styles isolated
3. Gathered team information and biographies
4. Setup team page skeleton with placeholder information and pictures

1. Created template and added usage for future reference

2. **Social Media Team**

1. Project teasing on instagram

1. Target location
2. Target Species
3. Microcystin vs *Microcystis* nomenclature

2. Thanked IMPACT grant sponsor
3. First educational critter o'clock post

3. **Dry Lab Team**

1. Refined a conjugative transposon approach for knocking-out microcystin production
2. Looked for shuttle vectors that have a selectable marker, replicate in *E. coli* and *M. aeruginosa*, and conjugate in *M. aeruginosa*
3. Drafted protocols for transforming *M. aeruginosa* based on the 2016 SSH iGEM team that succeeded in transforming *M. aeruginosa* with a dead Cas-9 that silenced the McyB gene essential for microcystin production
4. Made protocol for Bold 3N growth media
5. Met with Glenn Millhauser and Kevin Singewald about using the HPLC to measure microcystin levels

4. **Wet Lab Team**

1. Completed Inoue protocol for chemicompetent cells and evaluated transformation efficiency of PET28-GFP plasmid

1. Very low, restarted protocol from scratch

2. Received and started an *M. Aeruginosa* CPCC 300 strain (UTEX LB2385) culture

1. Transferred from original vial to mammalian cell culture flask for increased surface area

3. Started a second *M. Aeruginosa* culture using provided Bold 3N (B3N) media inoculated with 0.5ul of original *M. Aeruginosa* culture

## Week of 7/10/2023

1. **Fundraising Team**

1. Edited the grant applications
2. Reached out to potential sponsors

2. **Social Media Team**

1. IDT and Promega shoutout on social media
2. Social media story updates
3. Updated bio
4. Critter o'clock story updates

3. **Dry Lab Team**

1. Coordinated additional meeting with Kevin Singewald to learn how to use the HPLC
2. Drafted additional general protocols and HPLC based on personal protocols and literature
3. Introduction to Stealth bioinformatics tool to identify potential restriction motifs
4. Annotated pSPIN-R plasmid to determine all of the INTEGRATE components necessary for toxin disruption
5. Annotated pSHDY plasmid to determine all of the necessary components for broad host range conjugation
6. Started work on the Chameleon project

4. **Wet Lab Team**

1. Competent cells with high transformation efficiency

1. Top10 with pET28a

2. 1st time DNA extraction / troubleshooting

1. Low DNA yield, contamination

3. Received pSHDY plasmid from Addgene (Addgene plasmid # 137661)
4. Created 1% *M. aeruginosa* inoculation from initial culture

1. Done to keep planktonic cell culture

5. **Human Practices Team**

1. Presented to and completed learning activities with Girls in Engineering
2. Began reaching out to Pinto Lake authorities to identify researchers able to speak to us regarding the long-term considerations of our project

## Week of 7/17/2023

1. **Social Media**

1. IDT shoutout
2. Critter o'clock post
3. Instagram story updates
4. Promega shoutout

2. **Dry Lab Team**

1. Met with Kevin Singewald for an HPLC tutorial
2. Drafted a co-culturing protocol for *E. coli* and *M. aeruginosa*
3. Coordinated with the wet lab team to test parameters for co-culturing and troubleshooting
4. Designed early iteration of plasmid: a conjugative plasmid capable of toxin disruption

3. **Wet Lab Team**

1. Extracted *M. aeruginosa* DNA for sequencing

## Week of 7/24/2023

1. **Dry Lab Team**

1. Considered plasmid delivery mechanisms alternative to conjugation to bypass a helper plasmid requirement
2. Assessed Stealth for integration with our approach based on the horizontal propagation of our genetic construct
3. Wrote several small scripts to help with Stealth output processing
4. Assessed Stealth, a bioinformatics tool, for integration with our approach based on the horizontal propagation of our genetic construct.

1. Wrote several small scripts to help with Stealth output processing
2. Worked on streamlining use of Stealth via software pipeline

2. **Wet Lab Team**

1. Reattempted DNA extraction of *M. aeruginosa* DNA for sequencing

## Week of 7/31/2023

1. **Human Practices Team**

1. Met with Diego Gelsinger from Columbia University for advice on expression of CAST (CRISPR-associated transposons) systems and conjugation in prokaryotes.

2. **Dry Lab Team**

1. Considered alternative approaches for toxin disruption including:

1. Plasmid-mediated cell lysis, siRNA silencing, enzyme degradation, and insertional mutagenesis
2. Decided to pursue insertional mutagenesis after meeting with Diego Gelsinger from Columbia University

3. **Software Team**

1. Began development of a powerful software pipeline in order to enhance the potential and accessibility of the Stealth algorithm - Chameleon
2. Began converting previously written scripts into dynamic programs

4. **Wet Lab Team**

1. Interlab Calibration - measuring fluorescence

1. Did not use opaque plate, necessitating redoing experiment

2. Reattempted DNA extraction of M. aeruginosa DNA for sequencing
3. Prepared for nanopore sequencing and creation of a DNA library.

5. **Outreach Team**

1. Prepared for iGEM meetup

6. **Interlab Team**

1. Transformed DH5a cells with 8 provided interlab devices

### **August**

## Week of 8/7/2023

1. **Social Media Team**

1. Promega Shoutout
2. iGEM UCSC Meet-Up promotion / advertising
3. Critter o’clock

2. **Wet Lab Team**

1. Transformation of pSPIN and pSPIN-R into TOP10 cells
2. Successful pSHDY transformation into *M. aeruginosa*

3. **Sequencing Team**

1. Created DNA library
2. Loaded minION and ran it, stopped running due to power outage

4. **Interlab Team**

1. Repeated interlab experiment 3

1. DH5a transformation
2. Inoculation & Serial Dilutions
3. Plate measurements

5. **Dry Lab Team**

1. Drafted protocols for end-point conjugation between EcGT2 *E. coli* and *M. aeruginosa* based on Diego Gelsinger's *Nature protocols* preprint
2. Considered more foundational contributions to the iGEM community, like a software pipeline (Chameleon) that tailors plasmids to the RM profile of a specific bacteria

6. **Software Team**

1. Started development of a software pipeline centered around the Stealth program - Chameleon

7. **Outreach Team**

1. Presented at the iGEM meet up

## Week of 8/14/2023

1. **Wet Lab Team**

1. Miniprep and serial decade dilutions of pSHDY, transforming into M. aeruginosa

2. **Dry Lab Team**

1. Designed a broad-host range, conjugative plasmid (pSPDY) that we would use to validate Chameleon.

1. Planned to order two versions of pSPDY: one that was unmodified and another that was optimized by Chameleon

2. Designed gene blocks and flagged primers for synthesizing unmodified pSPDY with golden gate
3. Order flagged primers and gene blocks from IDT for synthesizing the unmodified pSPDY
4. Designed experiments for validating Chameleon through natural transformation and conjugation of pSPDY

3. **Sequencing Team**

1. Contacted Brandy McNulty (Miga Lab) and had assistance on loading the minION

1. Learned how to properly empty waste port
2. Learned how to wash and reload flow cell mid run

2. Ran the minION again for nanopore sequencing

4. **Software Team**

1. Explored alternative approaches to pattern constraint module in order to enhance cohesion between codon optimization and pattern avoidance activities
2. Brainstormed method to identify “mutable” regions of a plasmid to prevent modifications of non-coding sequence
3. Converted all Stealth output processing scripts to command-line programs
4. Drafted outline of the total pipeline including inputs, outputs, and run options

## Week of 8/21/2023

1. **Wet Lab Team**

1. Serial dilutions of pSHDY and transformation into M. aeruginosa
2. Conjugation with pSPIN delivery of *E. coli* EcGT2 and *M. aeruginosa*

2. **Interlab Team**

1. Repeat Interlab (used wrong plate previous run):

1. Switched to Experiment 2 due to lack of Experiment 3 devices
2. Transformation, Inoculation, Plate Read

3. **Dry Lab Team**

1. Worked closely with the software team to prepare Chameleon for modifying non-overlapping protein coding regions of pSPDY
2. Focused on revising our thesis

4. **Sequencing Team**

1. Looked at the results of previous sequencing and tried to differentiate the DNA between species

5. **Software Team**

1. Decided on GenBank record and FastA compatibility
2. Finished parsing mutable CDS regions from a plasmid
3. Test ran modules on pSPDY

6. **Outreach Team**

1. Reaching out to local schools and Watsonville Wetlands Watch
2. Met with the Santa Cruz County Water Quality Lab

## Week of 8/28/2023

- **Wet Lab Team**

1. Selection of transconjugants
2. Rehydrated primers for new plasmid
3. Ran PCR on pSPDY

- **Sequencing Team**

1. Created new DNA library

- **Software Team**

1. Modular testing of software pipeline, continued development
2. Brainstorm module-to-module integration to begin forming complete pipeline
3. Drafted project plan towards final submission

### **September**

## Week of 9/4/2023

1. **Wet Lab Team**

1. Performed natural transformation
2. Sequencing:

1. Fragments small -> Made clean DNA library and prepped
2. Reloaded flow cell

3. PCR and Golden Gate Assemble of pSPDY

2. **Dry Lab Team**

1. Worked closely with the software team to use Chameleon on pSPDY
2. Designed gene blocks for synthesizing modified pSPDY with golden gate
3. Order flagged primers and gene blocks from IDT for synthesizing modified pSPDY
4. Designed and edited promotional video

3. **Wiki Team**

1. Updating the notebook

1. Caught wiki up with back-log entries

2. Writing attributions and editing project descriptions
3. Drafted team logo and wiki colorscheme

4. **Software Team**

1. Developed Stealth output parsing algorithm to screen global underrepresented motifs down to workable level
2. Applied pipeline modules to the pSHDY plasmid file, creating fragments for IDT synthesis
3. Code Reformatting:

1. Each module can act as a standalone command line interface
2. Separate pipeline that uses all modules to seamlessly generate single output

## Week of 9/11/2023

1. **Software Team**

1. Command line interfaces reformatted to use new code structure
2. Drafted plan to integrate Stealth software into iGEM submission
3. Started work towards a Chameleon python package

1. Simple pip installation and command line interface use

2. **Wiki Team**

1. Began writing and organization for wiki writing. Assigned members to different pages of the wiki

3. **Wet Lab Team**

1. Transformations of Golden Gate product pSPDY unmodified
2. Golden Gate troubleshooting
3. Transferred microcystis cultures into new BG11 for cultivation

## Week of 9/18/2023

1. **Software Team**

1. Adopted a more modular package layout for pipeline
2. Integrated Stealth into package
3. Started building final pipeline

2. **Wet Lab Team**

1. Troubleshooting Golden Gate Assembly by running Colony PCR on suspected GG product
2. Drafting new ways to perform natural transformation in a liquid culture rather than a solid culture

3. **Outreach Team**

1. Gave a tour of our lab and a run down of our project to the Mayor of Santa Cruz, Fred Keely

## Week of 9/25/2023

1. **Software Team**

1. Renamed all submodules and reformatted code
2. Created a Stealth sub-class to encapsulate Stealth output into an object
3. Created a new codon analyzer
4. Published a working version of the pipeline

2. **Wet Lab Team**

1. Gene amplification of the Stealthed fragments for pSPDY modified
2. Golden Gate Assembly of pSPDY, still having some problems verifying unmodified pSPDY
3. Ethanol precipitation of pSPDY

### **October**

## Week of 10/2/2023

1. **Software Team**

1. Added code documentation

2. **Wet Lab Team**

1. Colony PCR of unmodified pSPDY transformed in *E. coli*
2. PCR amplification of gene fragments for modified pSPDY
3. Miniprep and sequencing of unmodified pSPDY

3. **Outreach Team**

1. Presented info presentation on our project and performed experiments with Mission Hill Middle School

## Week of 10/9/2023

1. **Wiki Team**

1. Finalization of wiki

2. **Wet Lab Team**

1. Colony PCR was performed in order to verify the magnitude of success of our Golden Gate assembly
2. Performed and troubleshooted Golden Gate Assembly of modified pSPDY including gel electrophoresis
3. Electroporation of unmodified pSPDY into *M. aeruginosa*
4. Gene fragment amplification of new iterations of fragments

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## Introduction

*Microcystis aeruginosa* is a naturally competent [1],
non-model cyanobacteria that is principally responsible for
toxic harmful algal blooms (HABs) in freshwater. *M. aeruginosa*
secretes microcystin, a potent hepatotoxin and allelopathic inhibitor
that threatens the utility and biodiversity of freshwater ecosystems globally
[2]. Initially, our team sought to leverage the natural competence of *M. aeruginosa*
to selectively disrupt the production of microcystin in transformed cells. However,
to disrupt microcystin production at the scale of a HAB, we recognized a greater
need to advance genetic tractability in non-model organisms like *M. aeruginosa*.
Restriction-modification (R-M) systems are a major barrier of the domestication
of non-model species due to the targeting of short nucleotide sequences by restriction
enzymes [3]. We believed that transformation efficiency in non-model organisms could
be improved by avoiding species-specific restriction sites during plasmid synthesis.
To this end, we developed the Chameleon project. Our work is built off of the Stealth
program [4], which produces putative restriction sites by identifying short nucleotide
sequences that are underrepresented in a specific organism's genome. Chameleon removes
putative restriction sites from protein-coding regions of plasmids through synonymous
codon-optimization. The following DBTL cycles were aimed at validating the use of
Chameleon in future bioengineering projects by transforming *M. aeruginosa* with modified and unmodified plasmids.

#### **Cycle 1**

##### **Transformation of *M. aeruginosa***

**Design:**
*M. aeruginosa* is naturally-competent [1]. Natural transformation involves localizing naked plasmid DNA and cells on a millipore filter over solid, nonselective media (see [Journal: Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)). After 1 day, the filter is transferred to solid, selective media to isolate natural transformants. This method does not form distinct colony forming units (CFU); instead, transformed cells form a lawn over the millipore filter. Our team was unable to quantify transformation efficiency by CFU/ug DNA plated with this method, so we designed a protocol based on serial plasmid dilutions. By plating constant cell densities with varying serial plasmid dilutions over each millipore filter, we believed we could quantify transformation efficiency on the basis of plasmid concentration needed to form a visible lawn. We reasoned that if transformation efficiency improved with a modified plasmid, then the modified plasmid should form a visible lawn at a lower concentration than the unmodified plasmid

**Build:**
For this purpose, we ordered pSHDY ([Addgene #137661](https://www.addgene.org/137661/)): a broad-host range, high-copy plasmid that has been engineered for expression in cyanobacteria. We facilitated three cycles of natural transformation with *M. aeruginosa* aimed at identifying the concentration of pSHDY that would not form a visible lawn. We based our stock pSHDY concentration (50 ng/ul) on a protocol devised by a former iGEM team ([SCCHK 2019](https://2019.igem.org/Team%3AHK_SSC)), and made twenty 1:10 serial dilutions of the stock in our final cycle.

**Test:**
We plated 100 uL of cells (OD730= 0.4) and 40 uL of plasmid on each membrane filter numbered 1-20 onto BG11 plates with 50 ug/ml Chloramphenicol (Fig. 1). Filter 1 was plated with 40 ul pSDHY stock, and filter 20 was plated with 40 ul of a 10-20 serial dilution of pSHDY stock. The plate labeled + control demonstrates the effect of plating 40 ul of stock with 100 ul or 10 ul of cells, while plate labeled - control demonstrates the effect of plating 100 ul or 10 ul of cells without plasmid.
![UTEX 2385 M. aeruginosa natural transformants with pSHDY on BG11+chloramphenicol plates](https://static.igem.wiki/teams/4592/wiki/engineering/eng.jpg)

**Figure 1.** UTEX 2385 *M. aeruginosa* natural transformants with pSHDY are grown on 0.25 um Millipore filters on BG11+chloramphenicol (50 ug/mL) plates. From filter 1 to 20 respectively: No dilution, 10-(1->20) dilution. The positive control with pSHDY and negative control with no plasmid.

**Learn:**
We observed a lawn of cells at every dilution of pSHDY at the time of taking this image, which was 10 days after transferring the filters to selective media. We recognized that a 10-20 dilution of pSHDY stock should not have yielded such a robust natural transformation of *M. aeruginosa*, and that the no plasmid control exhibited more growth than we'd expect on selective media. We attributed these results to the bacteriostatic mechanism of action for chloramphenicol [5]. Chloramphenicol inhibits bacterial growth rather than killing bacteria directly. The duration of the *M. aeruginosa* cell cycle is about 3-4 days, so we reasoned that chloramphenicol selection would not be effective for quantifying transformation efficiency by plating serial plasmid dilutions. After 3 natural transformation cycles, our team recognized that a serial plasmid dilution with chloramphenicol selection would be unviable for assaying transformation efficiency with modified and unmodified plasmids in *M. aeruginosa*, especially with respect to our project's time constraints. Moving forward, we decided to transform *M. aeruginosa* with electroporation because the protocol had been validated by a previous iGEM team (SCCHK 2019). This approach allows for chloramphenicol selection in liquid cultures, so we decided to quantify transformation efficiency by comparing hemocytometer counts of untransformed cultures to those transformed with modified and unmodified plasmid. We hypothesized that cultures transformed with modified plasmid would have greater hemocytometer counts than those transformed with unmodified plasmid.

Previous

Next

#### **Cycle 2**

##### **Nanopore MinION Genomic Sequencing**

**Design:**
In order to apply Stealth to *M. aeruginosa* and to generate a plasmid modified by the Chameleon project, it was necessary to sequence the genome of the UTEX 2385 *M. aeruginosa* strain. Several coding sequences of this strain had been published, but a complete genome was not available. To assemble a full genome, we designed protocols to extract and purify *M. aeruginosa* DNA to conduct Nanopore sequencing [6].

We decided to use a MinION device from Oxford Nanopore along with R9.4.1 flow cells and SQKLSK-112 DNA Library Prep Kit. Our initial materials and methodology were heavily influenced by available resources.

**Build:**

*DNA Extraction:*

To extract Microcystis DNA, it was necessary to lyse the cell without compromising the integrity of the DNA. The UTEX 2385 cell wall consists of peptidoglycan and lipopolysaccharide layers. We believed that enzymatic digestion of peptide bonds would be effective in gently lysing the cell without fragmenting the DNA. Following extraction, the DNA was purified through ethanol precipitation (see [Journal: Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D)).

*Sequencing:*

The SQKLSK-112 DNA Kit was used to prepare a library which was loaded onto two R9.4.1 flow cells. Due to reduced pore occupancy in one of our flow cells, we ran two MinION devices in parallel, aiming to obtain more comprehensive data for genome assembly. The minimum read length considered for both runs was set to 1000 bp. Reads were collected over 72 hours and we performed a wash and reload step after the first 24 hours to improve pore efficiency. Raw signals collected from the MinION were translated into nucleotide sequences using the base calling software Dorado 0.3.2 [7]

*Assembly:*

Following base-calling, we utilized Flye 2.9.2 [8] to assemble and align the reads, and Bandage 0.9.0 [9] to visualize the complete assembly.

**Test:**
To validate that the resulting genomic assembly represented *M. aeruginosa*, we used RNAmmer [10] to extract genes that encode 16s ribosomal RNA. We identified 11 contigs that contained 16s ribosomal RNA. Contigs that contained ribosomal RNA genes were then compared to sequences in the rRNA/ITS databases using NCBI BLASTN.

**Learn:**
Of the 11 contigs that contained ribosomal RNA genes, 3 had direct hits to *M. aeruginosa*. Interestingly, only 2 of these 3 hits made it into the Flye assembly; these are depicted in dark orange in Figure 1. The remaining contigs had hits to the genera *Gemmatimonas*, *Blastomonas*, *Hydrogenophaga* and *Stenotrophobacter*, which are colored in gray in Figure 1.
While we could verify the identity of 2 contigs depicted in the *M. aeruginosa* assembly, the full assembly was composed of 7 contigs. It was clear that we needed a definitive method to distinguish contigs that truly belonged to *M. aeruginosa* to ensure confidence in the sequence we used for the Stealth analysis.
Ultimately, this analysis confirmed that our UTEX 2385 culture was xenic, and that we successfully extracted *M. aeruginosa* DNA, however, our efforts only resulted in a partial assembly of the UTEX 2385 genome. By combining data from both runs, we assembled approximately 4 megabases from the expected 6 megabases of the *M. aeruginosa* genome using Flye [8] .
![Flye assembly visualized by Bandage](https://static.igem.wiki/teams/4592/wiki/engineering/eng2.png)

**Figure 1**. Flye assembly visualized by Bandage:
Colored contigs indicate correlation to the *M. aeruginosa*'s gDNA.

Previous

Next

#### **Cycle 3**

##### **Cluster-K**

**Design:**
To verify that the Nanopore-generated contig assembly belonged to *M. aeruginosa*, we utilized Cluster-K: a novel software tool written by our principal investigator that groups genetic sequences on a three-dimensional graph by considering their unique tetramer signature (Fig. 1).
Cluster-K samples all of the tetramers that exist in a genetic sequence and constructs a frequency matrix composed of 256 elements (44 elements). The frequency matrix generated for any given sequence is unique, and in some sense can be conceived as a genetic signature. In addition to the frequency matrix, Cluster-K considers GC content and codon usage of each tetramer to determine a sequence's signature, so signatures become more distinct as the length of the sequence increases.
From the 256 elements that describe a collection of sequences, Cluster-K will graphically depict three elements that have the greatest variance within a collection. The software employs Principal Component Analysis, a dimension reduction method based on the variance in the dimensional tree, and outputs a three-dimensional diagram with clustered contigs. These results can indicate whether the given group of contigs belongs to the same species.
![Cluster-K Output](https://static.igem.wiki/teams/4592/wiki/engineering/eng4.png)

**Figure 1** Cluster-K Output
Cluster 1 (in orange) predicts that elements of the cluster belong to the same species, *M. aeruginosa*

**Build:**
We employed Cluster-K to analyze the assembled contigs, resulting in the generation of a 3D graph that revealed eleven distinct clusters of correlated contigs (Figure 1).
![Cluster-K Output](https://static.igem.wiki/teams/4592/wiki/engineering/eng4.png)

**Figure 1** Cluster-K Output
Cluster 1 (in orange) predicts that elements of the cluster belong to the same species, *M. aeruginosa*

**Test:**
We compared Cluster-K output to that of the Bandage-visualized Flye assembly () to validate contigs that belong to the *M. aeruginosa* genome.

**Learn:**
We determined that the Cluster-K data reinforces the Bandage-visualized Flye assembly (Figure 1, 2) with contigs that are potentially a part of M.aeruginosa's gDNA and do not have a 16S rRNA, like contigs 10, 12, 18, 198, 199, 7 (Figure 3).
![Cluster-K Output](https://static.igem.wiki/teams/4592/wiki/engineering/eng4.png)

**Figure 1.** Cluster-K Output
Cluster 1 (in orange) predicts that elements of the cluster belong to the same species, *M. aeruginosa*

![Flye assembly visualized by Bandage](https://static.igem.wiki/teams/4592/wiki/engineering/eng3.png)

**Figure 2.** Flye assembly visualized by Bandage:
Colored contigs indicate correlation to the *M. aeruginosa's* genomic DNA.

![Venn diagram of BLASTN and Cluster-K](https://static.igem.wiki/teams/4592/wiki/engineering/eng5.png)

**Figure 3.** A Venn diagram of BLASTN and Cluster-K (group 1) output

Previous

Next

#### **Cycle 4**

##### **Construction of Modified and Unmodified Plasmids**

**Design:**
Starting from the pSHDY plasmid ([Addgene #137661](https://www.addgene.org/137661/)), we added an insert in *silico* containing the CaMV35S core promoter, the eGFP gene, the T7 terminator, and a RP4 mobilization gene; the insert is depicted in dark red in Figure 1. We named the new plasmid pSPDY and designed fragments to assemble two pSPDY constructs using Golden Gate: one that was modified by the Chameleon project to remove putative restriction sites (Fig. 2), and one that was unmodified (Fig.1). The modified and unmodified pSPDY plasmids were identical in all non-protein-coding regions. Protein-coding regions on the modified plasmid were codon-optimized by Chameleon for codon-usage in our *M. aeruginosa* UTEX 2385 partial genome assembly. Additionally, Chameleon removed putative, Stealth-identified restriction sites from protein-coding regions through synonymous codons optimization. This process necessitated that modified pSPDY be assembled from IDT genes blocks with Golden Gate, so we designed flagged primers to introduce ligation sites compatible with the PaqC1 Type IIS restriction enzyme. We also designed sequencing primers that would amplify across each ligation site to verify Golden Gate assembly.
![Unmodified pSPDY ](https://static.igem.wiki/teams/4592/wiki/engineering/eng6.png)

**Figure 1.** Unmodified pSPDY plasmid. The backbone of unmodified pSPDY is taken from the pSHDY plasmid. The insert is composed of the yellow, green, and blue Golden Gate fragments. The yellow fragment (159 bp) contains the CaMV35S core promoter (BBa\_K4592000) and associated RBS. The green fragment (1179 bp) contains the *Microcystis*-optimized eGFP gene (BBa\_K4592001) and the T7 terminator; therefore, the *Microcystis*-optimized cyanobacteria reporter (BBa\_K4592002) spans the yellow and green fragments. The blue fragment (1355 bp) contains mobilization genes that were previously intended to be used in conjugation experiments from *E. coli* to UTEX 2385 *M. aeruginosa*. Image made using Geneious Prime.

![Modified pSPDY plasmid](https://static.igem.wiki/teams/4592/wiki/engineering/eng7.png)

**Figure 2.** Modified pSPDY plasmid. The yellow, green, and blue Golden Gate fragments are identical to the unmodified pSPDY plasmid. Unlike the unmodified pSPDY plasmid, the modified pSPDY plasmid had to be mainly assembled from fragments in order to remove restriction sites from the original pSHDY sequences. The two gray fragments, pSHDY BB (small) and pSHDY BB (large), correspond to regions that were originally intended to be made from Golden Gate fragments; these fragments either could not be ordered due to unresolvable high complexity scores per IDT or were challenging to amplify and assemble with the other fragments; in these cases, amplicons of the pSHDY backbone prepared using the flagged primers intended for these regions' fragments were used to assemble the plasmid instead; although this entails the reintroduction of some restriction sites, it was a necessary accommodation to make progress in plasmid assembly. Image made using Geneious Prime.

**Build:**
gBlock Gene Fragments were ordered from IDT for each of the Golden Gate fragments to be assembled. Since these gene fragments did not include PaqC1 binding sites and fusion sites, they were first amplified with the flagged primers. The resulting amplicons were the Golden Gate fragments to be assembled in a Golden Gate reaction. Plasmids were assembled from these fragments by Golden Gate assembly, then transformed into TOP10 *E. coli* and plated on chloramphenicol selective media for replication before miniprep. Positive and negative controls were conducted alongside this process; the negative control was untransformed TOP10 *E. coli* and the positive control was TOP10 *E. coli* transformed with pSHDY, which confers resistance to chloramphenicol just as the pSPDY plasmids would and was already known to be functional in *E. coli*.

**Test:**
Successful putative pSPDY transformant *E. coli* were transferred to liquid cultures and viewed under a fluorescent microscope to check for eGFP expression. Checking for eGFP was necessary, particularly in the case of putative unmodified pSPDY transformants, because the original pSHDY plasmid was used as a template in one of the PCR reactions preceding Golden Gate assembly; it was therefore possible for observed transformants to have been pSHDY transformants rather than pSPDY transformants, necessitating distinguishing the two on the basis of the expression of eGFP, which would only occur in pSPDY transformants. To the same end, plasmid from putative pSPDY transformants was also miniprepped; this was both directly run on an agarose gel against pSHDY to compare plasmid sizes (as pSPDY was expected to be larger due to the insert) and used as the template in PCR reactions using the sequencing primers to verify each of the Golden Gate ligations.

**Learn:**
Successful Golden Gate assembly and transformation into TOP10 *E. coli* of our pSPDY plasmids required several attempts. Due to our controls, sequencing primers, agarose gels, fluorescent microscope, and submission of miniprepped plasmids for Sanger sequencing, we were able to identify the cause of most of these failures. In some cases, this was due to our cells not being competent, which was made evident by the absence of growth in the positive control. In other cases, this was due to the pSHDY plasmid being carried through PCR and Golden Gate assembly, being transformed into TOP10 *E. coli* instead of pSPDY; this was discovered via failing to observe fluorescence, the absence of expected amplicon bands in agarose gels following PCR with sequencing primers, and unexpectedly small plasmid bands on agarose gels of miniprepped plasmid. For unclear reasons, incorrect Golden Gate assembly occurred in at least one case wherein Sanger sequencing revealed the fragments to have assembled in an unintended order.

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#### **Cycle 5**

##### **Developing Chameleon Project**

**Design:**
The design phase of the Chameleon project was initiated after identifying the need for a solution to the barrier imposed by the restriction-modification systems, reaching past our project goals and into the goals of our field of research. At its core, the project is integrated with Stealth, a program developed and published by our PI, David Bernick. Stealth yields putative restriction sites that enhance transformation efficiency in non-model organisms if avoided in a genetic construct [4]. With respect to this advancement, we set out to develop an automated software pipeline that would take nothing more than a genomic sequence of the intended host (partial or complete) and a plasmid annotated in GenBank record to optimize transformation into the intended host. We prioritized efficiency, simplicity, and accessibility throughout software design.

**Build:**
To meet our aim, we identified a systematic approach toward plasmid optimization. Our pipeline starts with the Stealth program, which identifies underrepresented sequences in any given genome and saves those that are palindromic. (see [Software](https://2023.igem.wiki/ucsc/software)). The Chameleon project developed by our team predicts open reading frames (ORF’s) and derives a codon usage frequency profile from the host’s genomes. Concurrently, Chameleon analyzes the input GenBank record plasmid in order to identify the regions of the construct that may be altered; i.e non-overlapping protein-coding regions. This is a process we refer to as “unpacking”; this is an essential step in the pipeline in order to avoid introducing nucleotide changes that alter plasmid function. We recognized that codon usage is uniquely skewed in different organisms, so we remove putative restriction sites through stochastic, synonymous codon optimization with respect to the host’s endogenous codon usage. In this way, we ensure that we edit plasmids in a way that mirrors the codon frequency statistics of the host organism. Coding sequences must retain coding frames during substitution with synonymous codons. A sliding window approach is employed to achieve this, acting in conjunction with a method that generates every possible codon permutation. The window is sized to handle the maximum sized motif to be screened and no larger, preserves the stochastic usage of codons. Codon permutations are then filtered and collapsed based on the number of motifs present prior to further selection of optimal codon usage governed by usage frequencies, and deviation from the seed (which is minimized to maintain a codon distribution that reflects the hosts tRNA pool). The project was built to contain several proof-reading functions to ensure that computation and processing proceeds as intended.

**Test:**
A wide variety of test cases were employed throughout the build process, as the Chameleon project is the culmination of several key modules that perform discrete computation in an additive manner, requiring all components of the Chameleon project to work as intended on the individual level. To serve our project needs, the unmodified pSHDY backbone was used as input to the pipeline function of the Chameleon project to generate a modified pSPDY construct that contains a minimal number of motifs that Stealth identified as underrepresented. The Chameleon project successfully removed 76% of motif occurrences (Fig. 1 and 2) while optimizing codon usage and without altering the amino acid sequence (Fig. 3).
![Unmodified pSPDY](https://static.igem.wiki/teams/4592/wiki/engineering/eng8.png )

**Figure 1.** Unmodified pSPDY with putative restriction sites annotated. Each short pink annotation corresponds to a restriction site identified by Stealth that was matched to a sequence on the unmodified pSPDY plasmid. 197 putative restriction sites were identified on the unmodified pSPDY plasmid. Annotated regions colored in red were already passed through Chameleon to remove putative restriction sites. Annotated regions colored in yellow were not passed through Chameleon to remove putative restriction sites. Image made using Geneious Prime.

![Modified pSPDY](https://static.igem.wiki/teams/4592/wiki/engineering/eng9.png)

**Figure 2.** Modified pSPDY with putative restriction sites annotated. Each short pink annotation corresponds to a restriction site identified by Stealth that was matched to a sequence on the modified pSPDY plasmid. After processing of the unmodified plasmid with Chameleon, the majority of restriction sites on coding regions were removed without altering corresponding amino acid sequences. 40 putative restriction sites were retained on the modified pSPDY plasmid. Annotated regions colored in red were passed through Chameleon to remove putative restriction sites. Annotated regions colored in yellow were not passed through Chameleon to remove putative restriction sites. Image made using Geneious Prime.

![Comparison of chloramphenicol resistance gene](https://static.igem.wiki/teams/4592/wiki/c5fig3.png)

**Figure 3.** Comparison of chloramphenicol resistance gene (cat) on unmodified pSPDY (yellow) and modified pSPDY (red). Both versions of cat yield the same amino acid sequence despite different nucleotide sequences. There are 19 putative restriction sites identified on the unmodified cat CDS and 0 putative restriction sites identified on the modified cat CDS.

**Learn:**
Our initial design strategy led to several recursive instances of sub-DBTL cycles, and resulted in the Chameleon project functioning exactly as intended. The Chameleon project is able to produce a modified plasmid that contains a minimum number of Stealth identified palindromic motifs while retaining functionality and enhancing transformability [4]. Our initial design principles including modularity (to enable scalability and flexibility), generalizability (to impart a broad scope of applicability), and overall robustness allowed the structure and functionality of the pipeline to grow throughout the entire DBTL cycle. By the end of the cycle, we made the project available for anyone to conveniently download via pip, and have provided thorough documentation of the project. As an open source project, our source code is available for anyone to use. The TABI team plans to continue development of the Chameleon project post-Jamboree (2023).

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## References

1. F. Nies, M. Mielke, J. Pochert, and T. Lamparter, “Natural transformation of the filamentous cyanobacterium Phormidium lacuna,” PLOS ONE, vol. 15, no. 6, p. e0234440, Jun. 2020, doi: 10.1371/journal.pone.0234440.
2. R. Melaram, A. R. Newton, and J. Chafin, “Microcystin Contamination and Toxicity: Implications for Agriculture and Public Health,” Toxins, vol. 14, no. 5, p. 350, May 2022, doi: 10.3390/toxins14050350.
3. Q. Yan and S. S. Fong, “Challenges and Advances for Genetic Engineering of Non-model Bacteria and Uses in Consolidated Bioprocessing,” Front. Microbiol., vol. 8, p. 2060, Oct. 2017, doi: 10.3389/fmicb.2017.02060.
4. S. Hu, S. Giacopazzi, R. Giacopazzi, K. Karplus, D. Bernick, and K. Ottemann, “Altering under-represented DNA sequences elevates bacterial transformation efficiency.” University of California, Santa Cruz, Aug. 06, 2023.
5. J. J. Rahal and M. S. Simberkoff, “Bactericidal and Bacteriostatic Action of Chloramphenicol Against Meningeal Pathogens,” Antimicrob. Agents Chemother., vol. 16, no. 1, pp. 13-18, Jul. 1979, doi: 10.1128/AAC.16.1.13.
6. H. Lu, F. Giordano, and Z. Ning, “Oxford Nanopore MinION Sequencing and Genome Assembly,” Genomics Proteomics Bioinformatics, vol. 14, no. 5, pp. 265–279, Oct. 2016, doi: 10.1016/j.gpb.2016.05.004.
7. “Oxford Nanopore Technologies Dorado.” [Online]. Available: https://github.com/nanoporetech/dorado
8. B. Freire, S. Ladra, and J. R. Parama, “Memory-Efficient Assembly using Flye,” IEEE/ACM Trans. Comput. Biol. Bioinform., pp. 1–1, 2021, doi: 10.1109/TCBB.2021.3108843.
9. R. R. Wick, M. B. Schultz, J. Zobel, and K. E. Holt, “Bandage: interactive visualization of de novo genome assemblies,” Bioinformatics, vol. 31, no. 20, pp. 3350–3352, Oct. 2015, doi: 10.1093/bioinformatics/btv383.
10. K. Lagesen, P. Hallin, E. A. Rødland, H.-H. Stærfeldt, T. Rognes, and D. W. Ussery, “RNAmmer: consistent and rapid annotation of ribosomal RNA genes,” Nucleic Acids Res., vol. 35, no. 9, pp. 3100–3108, May 2007, doi: 10.1093/nar/gkm160

{% endblock %}

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{% block title %}PROTOCOLS{% endblock %}
{% block header\_image %} https://static.igem.wiki/teams/4592/wiki/banners/igem-2.jpg {% endblock %}
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## Colony PCR   Genotype bacteria without significantly disrupting culture

**Materials:**

* Bacterial Colonies
* Sterile Pipette Tips
* Additional Agar Plates
* One *Taq* Quick-Load 2X Master Mix
* Sterile H2O
* PCR Primer

**Equipment:**

* PCR Cycler

**Protocol:**

Colony PCR played a big role in troubleshooting and determining the efficacy of our golden gate assembly reactions following transformation. [NEB Robust Colony PCR Protocol (accessed 7/13/23)](https://www.neb.com/en-nz/-/media/nebus/files/application-notes/appnote_colony_pcr.pdf?rev=14eed5dce5764a2db77cf786f8c07f53) to fit our project needs, namely to accommodate liquid culture samples. Culture plate:

**1.** Transform ligation mix or other plasmid-con- taining reaction mixture into the desired bacterial strain, and incubate agar plates overnight at the appropriate temperature.

**2.** Set up 50 µl NEB OneTaq reactions consisting of 25μl 2x OneTaq master mix, 200nM primers, and add diH2O to top off to 50μl.

**3.** Use a sterile pipette tip to poke or pick up individual colonies and dip into each reaction tube. Generally speaking, the more the better (up to a colony or pellet with 2mm diameter), however merely poking colonies works when you do not wish to disrupt the colony, have good primers, and use touch-down PCR.

**4.** As soon as the solution looks cloudy, remove the pipette tip. To create a stock of each individual colony either: a.) Dip the pipette tip into 3 ml growth media with appropriate antibiotics and culture overnight. Or b.) Streak the pipette tip onto another agar plate containing the appropriate antibiotics and grow overnight.

**5.** Transfer reactions to a PCR cycler, and perform touch-down PCR within the NEB OneTaq guidelines.

**6.** Load 4–6 µl of each PCR reaction directly onto an agarose gel, alongside an appropriate DNA ladder.

## Touch-Down PCR   A variation of PCR with enhanced target specificity

We made frequent use of the Touch-Down PCR methodology in nearly every PCR experiment, and achieved significantly better results than with the traditional PCR methodology. Touch-Down PCR can be applied to many PCR kits, making sure to stay within the prescribed thermocycle parameters.

Touch-Down is a method for increasing specificity of PCR reactions and can be applied to many different PCR kits and is particularly useful when dealing with complex DNA samples or when you want to minimize nonspecific amplification. Touchdown PCR uses a cycling program where the annealing temperature is gradually reduced (e.g. 0.5-2°C /every second cycle). The initial annealing temperature should be several degrees above the estimated Tm of the primers. The annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued using this annealing temperature.

**Initial Denaturation:** This step is the same as in standard PCR, where the DNA is denatured at a high temperature.

**Touchdown Annealing:** In the initial 10 cycles, the annealing temperature is set 3°C higher than the actual Tm (melting temperature) of the primers, and stepped down 0.5°C each cycle reaching a final temp of 2 °C below the calculated annealing temp. This ensures that only highly specific binding occurs, reducing nonspecific binding or primer-dimer formation. In each subsequent cycle, the annealing temperature is lowered by a few degrees.

**Extension and Amplification:** The PCR reaction continues with primer extension and DNA amplification, as in standard PCR.

**Annealing:**  In the remaining ~20 cycles, annealing is continued at the lowest temp reached during touchdown (eg. 2°C below predicted annealing temperature)

**Extension and Amplification:** The PCR reaction continues with primer extension and DNA amplification, as in standard PCR.

**Final Annealing and Extension:** In the later cycles, the annealing temperature stabilizes at a temperature closer to the actual Tm of the primers, allowing for more efficient amplification of the target DNA.

The idea behind touchdown PCR is to start with stringent annealing conditions to enhance specificity and gradually transition to more permissive conditions to promote amplification of the target DNA. This helps reduce background noise and enhance the yield of the desired PCR product. Touchdown PCR can be particularly useful when dealing with templates that have a high degree of sequence similarity with non-target sequences or when you want to maximize the formation of difficult to amplify amplicons.

Protocol adapted from [NEB Touch Down PCR (accessed 7/20/23)](https://www.neb.com/en-us/faqs/0001/01/01/what-is-touchdown-pcr#:~:text=It%20is%20a%20method%20for,Tm%20of%20the%20primers)

## Preparation of Electrocompetent  *M. aeruginosa*   Achieves transformation more quickly than natural transformation

This protocol has been adapted from iGEM team HK SSC (2019) and Choi’s 2021 transformation protocols for cyanobacteria. While the protocol could benefit from larger culture volumes, we made use of smaller volumes to maximize variations of the protocol that we could attempt. Our target organism, *Microcystis aeruginosa*, is known to be both naturally and electro-competent, hence the pre-pulse incubation period with DNA.

**Materials:**

* *M.aeruginosa Culture*
* HEPES Buffer
* BG11
* Glucose
* Chloramphenicol

**Equipment:**

* Electroporator
* Centrifuge
* Microcentrifuge Tube

**1. Culturing Conditions:** Grow *Microcystis aeruginosa* cells at 25C under a 12/12 light cycle until they reach early log growth phase.

**2. Cell Collection:** Aliquot 10 mL of *Microcystis aeruginosa* culture into a centrifuge tube.

**3. Centrifugation:** Using a 4°C centrifuge, spin at 1000g for 12 minutes or until the supernatant becomes clear.

**4. Wash Steps:** Gently resuspend the cell pellets in 2.5 mL of 0.1 mM HEPES. Centrifuge at 1000g for 12 minutes or until the supernatant becomes clear. Repeat steps 3-4 for a total of three washes to reduce the concentration of extracellular salts and materials.

**5. Viability Check:** To assess cell viability, transfer 50uL of the cell suspension to a 0.2 mm electrotransformation cuvette. Perform electroporation with a pulse duration to achieve a time constant of ~5 msec. Follow the manufacturer's instructions for the specific electroporation system (e.g., Bio-Rad E. Coli Pulser). Repeat wash steps (3-4) if arcing occurs during electroporation.

**6. DNA Gradient:** Aliquot 50 uL of the cell suspension into micro-centrifuge tubes. Add 1 ng to 5 pg of the genetic construct in a gradient. This gradient approach allows for optimized DNA concentration to be utilized, maximizing transformation efficiency due to challenges in precise cyanobacteria cell count quantification.

**7. Pre-pulse Contact:** Transfer the tubes containing the cell suspension and DNA to ice and allow them to incubate for 1-5 hours.

**8. Electroporation:** Transfer 50 uL of each cell preparation to a sterile, chilled 0.2 mm electrotransformation cuvette. Deliver a 2.5 kV pulse according to the manufacturer's instructions (e.g., Bio-Rad E. Coli Pulser). Immediately after pulsing, add 1 mL of BG11 + 5mM Glucose medium to the cuvette and transfer the mixture to a culture tube (https://doi.org/10.3390/biom11020214, accessed 10/1/23).

**9. Incubation:** Transfer the culture tubes with cell suspension back to the original growth conditions (25C, 12/12 light cycle). Incubate for 24 hours. Note\* it is optimal for this step to be timed when growth lights are on.

**10. Antibiotic Selection:** Add 50 ug/mL chloramphenicol to the culture tubes. Incubate for approximately 2 days to allow antibiotic selection to take place.

**11. Transformation Efficiency Assay:**Assay transformation success and efficiencies as needed, considering the specific parameters and techniques relevant to your project.

## Natural Transformation of   *M. aeruginosa*   Transformation protocol

**Purpose:**  Test and analyze a plasmid's capability to transform our selected strain of *M. aeruginosa* through its capability of naturally uptaking foreign DNA.

**Materials:**

* *M.aeruginosa* Culture, grown in BG11
* 2% Agar BG11 Plates
* 10 mM NaCl
* Plasmid DNA
* Antibiotic
* 25mm diameter, 0.45 Millipore filters

**Equipment:**

* Incubator
* Biological Safety Cabinet

**1.** The natural transformation protocol was adapted from the 2019 HK\_SSC iGEM team. [24].

**2.** Pipet a sample of 30 mL of *M. aeruginosa* culture and centrifuge for 10 minutes at 2500 x g

**3.** Decant the supernatant and wash cells using 10 mM NaCl

**4.** Centrifuge again and a tenth of the original volume of BG-11 to resuspend the pellet

**5.** Pipet 2 µg of pSHDY into the sample and mix by inverting

**6.** Incubate the sample in the dark overnight at 20°C

**7.** The next day, add the culture to 25 mm diameter, 0.45 µM pore size millipore filters placed on BG-11 plates

**8.** Incubate the plates for 20 hours at 20°C in a 12:12 light dark cycle with a light lux level < 3200 and consistent gas exchange

**9.** Transfer the filters to 2% agar BG-11 plates containing antibiotics to select for transformants. The antibiotics used were spectinomycin, kanamycin and chloramphenicol at a 15µg/mL concentration.

**10.** Incubate the plates for 7-10 days at 20°C

## BG11 Media   BG11 media used to culture *Microcystis aeruginosa*

**Purpose:** BG11 media was made for the culturing and growth of *M. aeruginosa.* This media was selected due to its established efficacy in cultivating freshwater microalgae, marine microalgae, and cyanobacteria. The creation of this media as opposed to others is simpler and only requires materials more commonly found in the laboratory, relative to other options.

**Note:** \*KOD vitamins can be substituted for other supplements such as a vitamin B12+ biotin solution.

**Materials:**

* MilliQ water
* 11X Trace Metals solution
* 5M HCl
* 1000X KOD Vitamin\*
* Additional reagents listed in protocol

**Equipment:**

* 1 L glass bottle or flask
* Stir bar
* Magnetic stirrer
* pH meter
* Autoclave

**Protocol:**

**1.** Fill the glass bottle or flask to 900 mL with milliQ water.

**2.**Add 10 mL of each reagent listed sequentially:

* 17.6 mM NaNO3
* 0.23 mM K2HPO4
* 0.3 mM MgSO4·7H2O
* 0.24 mM CaCl2·2H2O
* 0.031 mM Citric Acid·H2O
* 0.021 mM Ferric Ammonium Citrate
* 0.0027 mM Na2EDTA·2H2O
* 0.19 mM Na2CO3

**3.**Add 1 mL of 1X Trace Metals solution to the flask.

**4.**Measure and adjust the pH of the solution to 7.5 using a pH meter and 5M HCl respectively.

**5.**To sterilize, place the bottle/flask in an autoclave and run a 30 minute liquid cycle.

**6.**After autoclaving, allow the solution to cool, add 10 µL 1000X KOD vitamins.

**7.**Store at 4°C.

## Golden Gate Assembly of pSPDY using PaqC1   Plasmid assembly

**Purpose:**  Golden Gate Assembly will be used to create stealthed and unstealthed versions of pSPDY with a promoter, fluorescence, and conjugation insert.

**Materials:**

* 2 microliters T4 DNA ligase buffer
* 2 microliters PaqC1
* 0.5 microliters PaqC1 activator
* 2 microliters T4 DNA ligase
* 1 microliter Destination plasmid (75 ng/microliter)

**Equipment:**

* Thermocycler
* PCR Tubes

The plasmid was assembled according to the PaqC1, T4 DNA ligase NEB [protocol](https://www.neb.com/en-us/protocols/2021/01/11/golden-gate-assembly-protocol-using-paqci-neb-r0745-and-t4-dna-ligase-neb-m0202)

**1.** Create a 20 µL reaction by combining the following in a PCR tube: 2 µL Hi T4 DNA Ligase 2 µL 10X T4 DNA Ligase Buffer 2 µL PaqC1 0.5 µL PaqC1 activator 75 ng of vector with 150 ng of each insert MilliQ water up to 20 µL

**2.** Run the reaction in the thermocycler under the following cycle parameters: 60 cycles at 37°C for 5 minutes, the 16°C for 5 minutes enzyme inactivation at 60°C for 5 minutes held at 4°C

**3.** The Golden Gate product was transformed into TOP10 E. Coli for replication, extracted with miniprep, and verified with PCR and gel electrophoresis to determine the successful plasmid assembly

## PCR Product Gel Electrophoresis  PCR verification

**Purpose:** Gel electrophoresis is employed to separate and visualize DNA fragments, enabling the assessment of PCR reaction success and the precise determination of fragment sizes. We utilized 0.8% agarose gel.

**Materials:**

* Agarose
* 1x TBE Buffer
* DNA Ladder
* DNA Sample
* Template DNA
* 20000X Apex DNA Safe Stain
* 6x Purple Loading Dye

**Equipment:**

* Casting tray
* Well Combs
* Voltage Source
* UV light source
* Microwave
* Analytical balance
* Weigh boat

**Protocol:**

**1.**Measure 0.8 g of agarose and add it to 100mL of 1X TBE Buffer.

**2.**Microwave solution in increments of 30 seconds until agar is fully dissolved. Swirl in between rounds.

**3.**Let the solution cool down to about 5°C.

**4.**Add 5 µL of 20,000X Apex DNA Safe Stain to the agar and swirl to mix.

**5.**Pour agar into casting tray with well comb, let it solidify.

**6.**Make the following sample mixes:

* 5 µL of DNA sample
* 1 µL of 6X Purple Loading Dye

**7.**Load sample and DNA ladder into wells.

**8.**Run gel at a voltage of 120V.

**9.**Stop the gel run when the loading dye is around 80% down the gel.

**10.**Visualize with gel imaging system.

## DNA Isolation from   *Microcystis aeruginosa*  DNA extraction

**Note:**  Protocol adapted from  [Zymo Research's](%E2%80%9D) Quick DNA HMW MagBead Kit

**Appendix A: Microbial Lysis**

**1.**  Pipette 800 µL of *M.aeruginosa* culture, and centrifuge at 7000 rcf for 6-8 minutes or until the pellet forms.

**2.** Remove supernatant and set aside on ice.

**3.** Add 100 µL 1X PBS buffer to the pellet and resuspend with a vortexer.

**4.** Centrifuge at 5000 rcf for 1 minute.

**5.** Remove supernatant and combine supernatant with leftover supernatant from step 2

**6.** Add 1 mL PBS to the pellet and resuspend.

**7.** Centrifuge at 5000 rcf for 1 minute, save the pellet and discard the supernatant. Add 100 µL of TE buffer to the pellet (centrifuge) and resuspend with TE buffer.

**8.** Add 20 µL of 10% SDS to the pellet.

**9.** Incubate between 60°C - 80°C in a heat block for 10 minutes.

**10.** Add saved supernatant from step 2 to the incubated sample.

**11.** Add 10 µL of Proteinase K (pipette mix 10X) to the sample.

**12.** Incubate at 55°C for 10 minutes on a heat block.

**13.** Centrifuge at 5000 rcf for 1 minute or until a pellet forms. Record the supernatant volume and discard the supernatant.

**14.** Add equivalent amounts of MilliQ water to replace the supernatant.

**15.** Add 800 µL of MagBinding Buffer and mix well.

**Appendix B: DNA Binding and Extraction**

**1.** Add 400 µL of Quick-DNA MagBinding Buffer to 400 µL of *Microcystis aeruginosa* sample. Mix 5 times by pipetting up and down.

**2.** Add 33 µL of MagBinding Beads to the solution. Mix 5 times by pipetting up and down.

**3.** Secure the sample on a vortexer and vortex for 10 minutes at a medium-intensity setting.

**4.** Transfer to a magstand, remove and discard the supernatant.

**5.** Add 500 µL MagBinding Buffer. Mix by pipetting up and down 5 times to resuspend.

**6,** Vortex at setting 2 for 5 minutes.

**7.** Transfer to magstand, remove and discard supernatant.

**8.** Add 500 µL of DNA pre-wash buffer. Pipette up and down 10 times to mix.

**9.** Transfer to the magstand, remove the supernatant and discard.

**10.** Add 900 µL of gDNA wash buffer, and mix by pipetting up and down 10 times.

**11.** Transfer all liquid and beats to a new microcentrifuge tube.

**12.** Transfer to a magstand, remove and discard supernatant.

**13.** Repeat steps 10-12.

**14.** Dry beads on a heat block set to 55°C for 10 minutes.

**15.** Add 58 µL DNA elution buffer. Place on the vortex for 5 minutes at room temperature.

**16.** Transfer for magstand, remove supernatant, and place into a new microcentrifuge tube.

**17.** Store at -20°C.

## Long-Read Nanopore Sequencing  Sequencing of UTEX 2385

**Purpose:** This process was used to prepare the genomic DNA of *M. aeruginosa* UTEX 2385 for genomic sequencing with Oxford Nanopore MinION. The results were then utilized by the Stealth program to find underrepresented sequence motifs.

* DNA Library Preparation adapted from archived SQKLSK112, Oxford Nanopore Technologies

**Materials:**

* MinION real-time sequencing device
* R9.4.1 flow cell
* Heat block
* Incubator
* Microcentrifuge
* Heat gun
* Hula mixer (gentle rotator mixer)
* Magnetic separator, suitable for 1.5 ml Eppendorf tubes
* Vortex mixer
* Thermal cycler
* P1000, P200, P100, P20, P10, P2 pipette and tips
* Ice bucket with ice
* Timer
* Qubit fluorometer (or equivalent for QC check)

**Equipment:**

* Ligation Sequencing Kit (SQK-LSK112)
* 1.5 ml Eppendorf DNA LoBind tubes
* 0.2 ml thin-walled PCR tubes
* Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
* Freshly prepared 70% ethanol in nuclease-free water
* QubitTM Assay Tubes (Invitrogen, Q32856)
* Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)

**DNA Library Preparation**

**1.** Thaw the DNA Control Sample (DCS) at room temperature, spin it down, mix by pipetting, and place it on ice.

**2.** Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with the manufacturer’s instructions, and place them on ice.

* Thaw all the reagents on ice.
* Flick and/or invert the reagent tubes to ensure they are well mixed.
* Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.
* Always spin down tubes before opening for the first time each day.
* The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilize any precipitate.
* The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

**3.** Prepare the DNA in nuclease-free water:

* Transfer 1 μg (or 100-200 fmol) genomic DNA into a 1.5 ml Eppendorf DNA LoBind tube.
* Adjust the volume to 47 μl with nuclease-free water.
* Mix thoroughly by pipetting up and down, or by flicking the tube.
* Spin down briefly in a microfuge.

**4.** In a 0.2 ml thin-walled PCR tube, mix the following:

* 47 µL of DNA from previous step
* 1 µL of DNA CS (optional)
* 3.5 µL of NEBNext FFPE DNA Repair Buffer
* 2 µL of NEBNext FFPE DNE Repair Mix
* 3.5 µL of Ultra II End-prep Reaction Buffer
* 3 µL of Ultra II End-prep Enzyme Mix
* **Total:**  60 µL

Between each addition, pipette mic 10-20 times

**5.** Ensure the reaction is thoroughly mixed by gently pipetting and spin down briefly.

**6.** Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.

**7.** Resuspend the AMPure XP Beads (AXP) by vortexing.

**8.** Transfer the DNA sample to clean 1.5ml Eppendorf DNA LoBind tube.

**9.** Add 60 µL of resuspended AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.

**10.** Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

**11.** Prepare 500 µL of fresh 70% ethanol in nuclease-free water.

**12.** Spin down the sample and pellet on a magnet until the supernatant is clear and colorless. Keep the tube on the magnet, and pipette off the supernatant.

**13.** Keep the tube on the magnet and wash the beads with 200 μl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

**14.**  Repeat the previous step.

**15.** Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

**16.** Remove the tube from the magnetic rack and resuspend the pellet in 61 μl nuclease-free water. Incubate for 2 minutes at room temperature.

**17.** Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.

**18.** Remove and retain 61 μl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

**Adpater Ligation and Clean-Up**

**1.** Spin down the Adapter Mix H (AMX H) and Quick T4 Ligase, and place on ice.

**2.** Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.

**3.** Thaw the Elution Buffer (EB) at room temperature, mix by vortexing, spin down, and place on ice.

**4.** To enrich DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down, and place on ice.

**5.** To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down, and place on ice.

**6.** In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:

* 60 µL of DNA sample from the previous step
* 25 µL of Ligration Buffer (LNB)
* 10 µL of NEDNext Quick T4 DNA Ligase
* 5 µL of Adapter Mix H (AMX H)
* **Total:**  100 µL

**7.** Ensure the reaction is thoroughly mixed by gently pipetting and spin down briefly.

**8.** Incubate the reaction for 10 minutes at room temperature.

**9.** Resuspend the AMPure XP Beads (AXP) by vortexing.

**10.** Add 40 μl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.

**11.** Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.

**12.** Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

**13.** Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

**14.** Repeat the previous step.

**15.** Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

**16.** Remove the tube from the magnetic rack and resuspend the pellet in 15 μl Elution Buffer (EB). Spin down and incubate for 10 minutes at room temperature. For high molecular weight DNA, incubating at 37°C can improve the recovery of long fragments.

**17.** Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.

**18.** Remove and retain 15 µl of eluate containing the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube. Dispose of the pelleted beads. Quantify 1 µl of eluted sample using a Qubit fluorometer.

The prepared library is used for loading into the flow cell. Store the library on ice until ready to load.

## Culturing  *M. aeruginosa*   Preparing samples

**Materials:**

* BG11 media
* Microcystis cells or culture

**Equipment:**

* Culture flasks that allow for air exchange
* Incubator and shaker with reflective cover, or growth chamber
* Light with timer

**Protocol:**

**1.** Inoculate 300 uL of microcystis culture into 30 mL of BG11 media.\*

**2.** Incubate at 25° C for optimal growth or at 20° C for optimal microcystin production at <3200 lux light, a 12:12 hour light/dark cycle, and consistent shaking and gas exchange.

**3.** \*Transfer of microcystis culture should be done in a BSL-2 cabinet or setting.

## PCR of gene fragments  Preparing golden gate fragments

**Purpose:** Assembling and amplifying gene fragments of unmodified and modified pSPDY plasmids.

* PCR using Q5® High-Fidelity DNA Polymerase was performed.
* Adapted from New England Biolabs’ PCR Using Q5® [High-Fidelity DNA Polymerase protocol](https://www.neb.com/en-us/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491)

**Materials:**

* 5X Q5 Reaction Buffer\*
* 10 mM dNTPs
* 10 µM Forward primer
* 10 µM Reverse primer
* Template DNA
* Q5 High-Fidelity DNA Polymerase\*
* Nuclease-Free water

**Equipment:**

* PCR Tubes
* Thermocycler

**Protocol:**

**1.** Create a 50 µL reaction by combining the following in a PCR tube:

* 10 µL 5X Q5 Reaction Buffer
* 1 µL 10 mM dNTPs
* 2.5 µL 10 µM Forward Primer
* 2.5 µL 10 µM Reverse Primer
* 1000 ng Template DNA
* 0.5 µL Q5 High-Fidelity DNA Polymerase
* Fill up to 50 µL Nuclease-free Water

**2.** Place the PCR reaction in the thermocycler and run a touchdown PCR protocol:

* Initial Denaturation: 98°C for 30 seconds
* 25 Cycles of the following:

+ 98°C for 10 seconds
+ Annealing: apply a touchdown method (3°C above and 2°C below the melting point of the primers used) for 30 seconds
+ 72°C for 30 seconds/kb

* Final Extension: 72°C for 2 minutes
* Hold at 4°C

\*Other methods and reagents for PCR may be used in place of Q5.

## Primer and Loading the Spoton Flow Cell  MinION sequencing

**Protocol:**

**1.** Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT), and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing and spin down at room temperature.

**2.** To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.

**3.** Open the MinION lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.

**4.** Slide the flow cell priming port cover clockwise to open the priming port.

**5.** After opening the priming port, check for a small air bubble under the cover. Drawback a small volume to remove any bubbles:

**a.** Set a P1000 to 200 µL

**b.** Insert the tip into the priming port.

**c.** Turn the wheel until the dial shows 220-230 ul, to draw back 20-30 ul, or until you can see a small volume of buffer entering the pipette tip.

**6.**  Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 minutes. During this time, prepare the library for loading by following the steps below.

**7.** Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting. The Loading Beads II (LBII) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

**8.** In a new tube, prepare the library for loading as follows:

* 37.5 µL of Sequencing Buffer II (SBII)
* 25.5 µL of Loading Beads II (LBII) mixed immediately before use, or Loading Solution (LS), if using
* 12 µL of DNA Library

**Total:**  75 µL

**9.** Complete the flow cell priming:

**a.** Gently lift the SpotON sample port cover to make the SpotON sample port accessible.

**b.** Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

**10.**  Mix the prepared library gently by pipetting up and down just prior to loading.

**11.** Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

**12.** Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

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#### Summary of Results

Our project was primarily successful in its bioinformatic and sequencing aspects. We have successfully sequenced the bidirectional promoter regulating the genes responsible for microcystin synthesis in UTEX 2385 *M.aeruginosa*, enabling specific targeting of the mcy cluster to halt microcystin toxin production in the future. We have also successfully prepared a partial assembly of the UTEX 2385 *M. aeruginosa* genome, validating it using Cluster-K (see Engineering). Our Chameleon software project has been completed and validated via its ability to remove restriction sites on DNA sequences in silico. A consensus RBS for UTEX 2385 *M. aeruginosa* has been identified and strength-assessed using bioinformatic techniques and made available on the iGEM Parts Registry. Fragments for Golden Gate assembly of the modified and unmodified pSPDY plasmids were prepared via amplification of gBlock Gene Fragments with flagged primers and verified.

---

#### Sequencing *mcy* gene cluster promoter region

In order to silence expression of downstream microcystin synthetase genes in our future integrative plasmid, we needed to sequence the bidirectional promoter of the mcy cluster in UTEX 2385 *M.aeruginosa* [1]. Partial CDSs of the synthetase genes and parts of the promoter region have previously been sequenced in UTEX 2385 and other *M. aeruginosa* strains, however the region spanning from mcyD to mcyA has not been previously sequenced in this strain. Since most of the region was unsequenced before our work, designing primers to generate an amplicon covering the region presented a challenge; by using regions of the mcyD and mcyA partial CDSs from other strains, we were able to design two primers that would bind to the genes flanking the promoter region. Using these primers in a PCR reaction with UTEX 2385 *M. aeruginosa* genomic DNA as template, an amplicon of the promoter region was created. The amplicon and primers were then sent out to the UC Berkeley DNA Sequencing Facility for Sanger sequencing; the resulting chromatogram was analyzed to produce the nucleotide sequence of the mcy cluster promoter region.

![](https://static.igem.wiki/teams/4592/wiki/annotated-sequence.png)

Figure 1. Annotated consensus sequence of a 1,131 BP region covering part of the mcyD gene, the bidirectional promoter region, and part of the mcyA gene of *M.aeruginosa* UTEX 2385. Annotations were made based on alignment to sequences in the published *M.aeruginosa* PCC 7806 mcy cluster. Image rendered in Geneious Prime.

---

#### Partial sequencing and Cluster-K validation of *M. aeruginosa* genome

![](https://static.igem.wiki/teams/4592/wiki/bandage.png)

Figure 2. Flye assembly visualized by Bandage: Colored contigs indicate correlation to the *M.aeruginosa*'s genomic DNA.

To implement the Stealth technique on *M.aeruginosa* and create a plasmid modified by the Chameleon project, we initiated the process by sequencing the genome of the UTEX 2385 *M. aeruginosa* strain using the MinION platform from ONT. Subsequently, a substantial amount of genomic DNA, approximately 4 out of 6 megabases, was successfully assembled through Flye and visualized with Bandage, as depicted in Figure 2.

The assembled data underwent further processing to extract the 16S rRNA sequences. These sequences were then subjected to a Nucleotide BLAST analysis, which revealed that only three contigs (namely contig 17, 2, and 8) were confirmed to belong to *M. aeruginosa*. Given the complex nature of our culture, it is important to note that *M.aeruginosa* exists as a xenic culture, meaning it coexists with various other bacterial species. To ensure the accuracy of our findings, an additional verification step was deemed necessary.

To address this, we employed Cluster-K, a tool that not only supported the 16S rRNA data but also introduced potential candidate contigs that might be associated with *M.aeruginosa* (Figure 3, 4). It is worth emphasizing that Cluster K's utility extends beyond our specific case, particularly in metagenomic analyses and environmental samples where the diversity of species to be analyzed is considerably higher.

For more details on Cluster-K see the [Engineering Page](https://2023.igem.wiki/ucsc/engineering) cycle 3

![](https://static.igem.wiki/teams/4592/wiki/cluster-k.png)

Figure 3 Cluster-K Output: Cluster 1 (in orange) predicts that elements of the cluster belong to the same species,*M. aeruginosa*

![](https://static.igem.wiki/teams/4592/wiki/venn.png)

Figure 4. A Venn diagram of BLASTN and Cluster-K (group 1) output

---

#### Design of Synthetic *M. aeruginosa* RBS

Using the Salis RBS Library Calculator v2.1.1, a synthetic RBS with a high predicted translation initiation rate (TIR) for *M.aeruginosa* was generated [2], [3], [4]. Generating the RBS required a CDS sequence and specified organism; the sequence of our eGFP gene optimized for expression in UTEX 2385 *M. aeruginosa*(documented on the BBa\_K4592001 page on the iGEM Parts Registry) was input with PCC 7806 *M. aeruginosa* (the closest organism available on the database). In the generated library, the synthetic RBS with the highest predicted translation initiation rate was AAGGAGG with a translation initiation rate of 817373.40 au; this RBS is documented on the BBa\_K4592003 page on the iGEM Parts Registry. We plan to use this synthetic RBS to express the eGFP reporter in UTEX 2385 *M.aeruginosa* with high efficiency, allowing us to validate transformations more easily. In doing so, we will also be able to empirically assess the strength of the synthetic RBS based on resulting eGFP fluorescence.

![](https://static.igem.wiki/teams/4592/wiki/table-sm.png)

Table 1. Features of synthetic RBS (BBa\_K4592003) predicted by Salis RBS Library Calculator v2.1.1.

---

#### Preparation of unmodified and modified pSPDY assembly via gene fragment amplification

gBlock Gene Fragments for the unmodified and modified pSPDY assemblies were amplified with flagged primers, introducing PaqC1 binding sites and fusion sites for Golden Gate assembly. To verify the success of this step, the resulting Golden Gate fragments were run on an agarose gel and compared to their expected sizes based on their in silico design. We verified the amplification of IDT gene blocks and addition of Golden Gate fusion sites of fragments with their expected amplicon band size. However, nonspecific amplification of gene fragments blue and ultraviolet was apparent. Amplification of the lime fragment, an assembly of the pSHDY backbone, yellow fragment, and green fragment, was not present on the gel, but is not necessary for assembly (Figure 5). For information on function and association of the color-coded Golden Gate fragments, see the section on plasmid construction in Engineering.

![](https://static.igem.wiki/teams/4592/wiki/gel.png)

Figure 5. A 0.8% agarose gel depicting the PCR products of amplified IDT gene fragments used for golden gate assembly of unmodified and modified pSPDY plasmid. The gene fragments were amplified with added flag primers that inserted Golden Gate fusion sites during the PCR cycle.

![](https://static.igem.wiki/teams/4592/wiki/table-b.png)

Table 2.Gel content for fragment amplification verification and expected gene fragment sizes.

---

#### Testing the Chameleon Project

The unmodified pSPDY construct contains many restriction sites, while the modified pSPDY construct has been passed through Chameleon. Chameleon removed most of the putative restriction sites that were identified by the Stealth program through synonymous codon-optimization of protein-coding sequences. The sequences that are modified by Chameleon on both plasmids are depicted in red in Figures 6 and 7. All other sequences are not protein-coding, or are overlapping protein coding regions that could not be altered through synonymous codon-optimization. The Chameleon project will only consider non-overlapping protein-coding regions for restriction site avoidance to maintain the functionality of any plasmid. The Chameleon project only requires a partial genome sequence to optimize any plasmid sequence in GenBank format. This design functions as a generic tool for increasing the transformability of any GenBank plasmid, in any partially-sequenced species.

To validate the function of the Chameleon software, we annotated all of the putative restriction sites identified by the Stealth program on both modified and unmodified pSPDY. The sequences annotated in pink in Figures 6 and 7 were underrepresented in the partial genome we assembled for the UTEX 2385 *M.aeruginosa*> strain, and are therefore treated as putative restriction sites by the Chameleon project. The unmodified plasmid exhibited 197 total putative restriction sites, while the modified plasmid only exhibited 40. The Chameleon project successfully removed 76% of all palindromic, putative restriction sites identified by Stealth without altering any corresponding amino acid sequences (Fig. 8). These results demonstrate success of the Chameleon project bioinformatically.

![](https://static.igem.wiki/teams/4592/wiki/plasmid-1.png)

Figure 6. Unmodified pSPDY with putative restriction sites annotated. Each short pink annotation corresponds to a restriction site identified by Stealth that was matched to a sequence on the unmodified pSPDY plasmid. 197 putative restriction sites were identified on the unmodified pSPDY plasmid. Annotated regions colored in red were already passed through Chameleon to remove putative restriction sites. Annotated regions colored in yellow were not passed through Chameleon to remove putative restriction sites. Image made using Geneious Prime.

![](https://static.igem.wiki/teams/4592/wiki/plasmid-2.png)

Figure 7. Modified pSPDY with putative restriction sites annotated. Each short pink annotation corresponds to a restriction site identified by Stealth that was matched to a sequence on the modified pSPDY plasmid. After processing of the unmodified plasmid with Chameleon, the majority of restriction sites on coding regions were removed without altering corresponding amino acid sequences. 40 putative restriction sites were retained on the modified pSPDY plasmid. Annotated regions colored in red were passed through Chameleon to remove putative restriction sites. Annotated regions colored in yellow were not passed through Chameleon to remove putative restriction sites. Image made using Geneious Prime.

![](https://static.igem.wiki/teams/4592/wiki/sequence-2.png)

Figure 8. Comparison of chloramphenicol resistance gene (cat) on unmodified pSPDY (yellow) and modified pSPDY (red). Both versions of cat yield the same amino acid sequence despite different nucleotide sequences. There are 19 putative restriction sites identified on the unmodified cat CDS and 0 putative restriction sites identified on the modified cat CDS.

---

#### Future Plans

Due to time constraints, we were unable to complete much of the wet lab work that would validate our dry lab work. We are continuing to work on the full sequencing of the UTEX 2385 *M.aeruginosa* genome, validating assembly of both the modified and unmodified pSPDY plasmids, comparing transformation efficiency in UTEX 2385 *M.aeruginosa* between the two plasmids to provide empirical validation of our Chameleon software project, characterizing expression mediated by the CaMV35S core promoter in UTEX 2385 *M.aeruginosa*> , and empirically validating our synthetic RBS. During the wiki thaw and before the re-freeze, we will add updates on our progress on these goals

Beyond the initial scope of this iGEM project, we also intend to make use of the improvement in transformation efficiency in UTEX 2385 *M.aeruginosa* to make a high transformation efficiency plasmid to knock out expression of microcystin-synthesizing genes in *M. aeruginosa*, enabling us to effectively address its harmful algal blooms.

---

#### Considerations for Replication

If our experiments were to be replicated, we have several considerations to share with experimenters:

* As both the Stealth program upon which our Chameleon project is built and the Chameleon project itself are continuing to be updated beyond this iGEM project, the outputs produced by the most recent versions may differ significantly from what is reported in our project even if the same inputs are used; experimenters may therefore consider using the static version of the Chameleon project hosted on the iGEM software Gitlab that corresponds to what was used for this project.
* Our work in transforming both E. coli and UTEX 2385 *M.aeruginosa* with our plasmids was hampered by some of our early design decisions: namely, the use of chloramphenicol as a selection marker, the composition of our insert, and the sequences of our gBlock Gene Fragments.

+ Chloramphenicol presented challenges for the selection of transformants in that it is bacteriostatic rather than bacteriocidal; since *M.aeruginosa*> has a slow doubling time, it was difficult to distinguish between untransformed cells whose ribosomes were inhibited by chloramphenicol and transformed cells that were simply replicating slowly.
+ Our insert included a reporter (eGFP), but not the chloramphenicol resistance gene; this resistance gene was already on the pSHDY backbone, which meant that additional protocols were needed to determine whether observed transformants had taken up the intended pSPDY construct or pSHDY that had persisted through the PCR amplification and Golden Gate assembly. Experimenters should consider having different resistance genes on the backbone and insert such that transformants that took up the new plasmid can be separated from transformants that took up the old plasmid.
+ Our gBlock Gene Fragments were designed as the sequences that would exist in the assembled plasmid. As a result, flagged primers adding PaqC1 recognition sites and fusion sites to the PCR amplicon were required. These created unnecessary potential points of failure in that the flagged primers had more opportunities to dimerize or bind to off-target regions, yielding Golden Gate fragments that would lead to an incorrect plasmid assembly. Since the gBlock Gene Fragments are synthesized de novo, we advise experimenters to design the fragments to already include PaqC1 (or the preferred Type IIS restriction enzyme) recognition sites and fusion sites.

**References**

[1] M. Kaebernick, E. Dittmann, T. Börner, and B. A. Neilan, “Multiple Alternate Transcripts Direct the Biosynthesis of Microcystin, a Cyanobacterial,” Appl. Environ. Microbiol., vol. 68, no. 2, pp. 449–455, Feb. 2002, doi: 10.1128/AEM.68.2.449-455.2002.

[2] A. C. Reis and H. M. Salis, “An Automated Model Test System for Systematic Development and Improvement of Gene Expression Models,” ACS Synth. Biol., vol. 9, no. 11, pp. 3145–3156, Nov. 2020, doi: 10.1021/acssynbio.0c00394.

[3] I. Farasat, M. Kushwaha, J. Collens, M. Easterbrook, M. Guido, and H. M. Salis, “Efficient search, mapping, and optimization of multi‐protein genetic systems in diverse bacteria,” Mol. Syst. Biol., vol. 10, no. 6, p. 731, Jun. 2014, doi: 10.15252/msb.20134955.

[4] C. Y. Ng, I. Farasat, C. D. Maranas, and H. M. Salis, “Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration,” Metab. Eng., vol. 29, pp. 86–96, May 2015, doi: 10.1016/j.ymben.2015.03.001.

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