Suicide Vectors for allelic exchange in Cellulophaga Lytica

By Mehul Puri

Introduction 

C. Lytica 

The gram-negative marine-based bacteria C. lytica has a genome of 3,765,936 base pairs, including 3,303 protein-coding genes and 55 RNA genes (Pati et al., 2011). It can grow in a wide range of temperatures between 4 °C and 40 °C in an 8% NaCl concentration with optimal growth between 22 °C to 30 °C (Pati et al., 2011). In the absence of flagella and pili, C. lytica cells depend on gliding motility to transport themselves. This translocation mechanism is also used to form biofilm colonies, which consist of colonies of C. lytica cells that can grow on non- biological surfaces such as rocks and metals. This allows them to survive in hostile environments and colonize new environments with ease. (Hall-Stoodley et al., 2004). 

Additionally, biofilm colonies serve as a foundation for larvae growth due to chemical and physical cues (Unabia et al., 1999). The formation of biofilm colonies by C. lytica also produces iridescence or coloration created by light reflection on intricately organized cells resembling crystals, as shown in Figure 1 (DeSimone, 2021). Though this iridescence has not been observed in natural environments, it has been observed in colonies grown in lab environments, and the significance of this iridescence in nature has not been elucidated to date. It is one of the identifiers or markers of biofilm colonies for C. lytica cells (Kientz et al., 2016). 

Figure 1. Colonies of C. lytica grown on Black ink plates (Adapted from M. DeSimone’s thesis, DeSimone 2021) 

Goal 

This study hypothesizes that deletion of the GldB gene in the bacteria Cellulophaga Lytica is responsible for gliding motility and can disrupt the formation of colonies. The ability to disrupt the formation of uniform colonies of C. lytica can impact the biofilm formation and allow us to control the iridescence of the bacteria. 

Approach/Methodology 

Designing the Vector 

In this project, the PYT313 suicide vector (Donated by a collaborator’s lab, Dr. Yontao Zhu, Minnesota State University Mankato) was used as it works with F. johnsoniae related to C. lytica. The suicide vector, as shown in Figure 2, contains sacB and the promoter of F. johnsoniae, ompA, which is used to construct chromosomal gene deletions specific to gliding (Zhu, 2017). Additionally, PYT313 is resistant to the antibiotic ampicillin due to the presence of AmpR. 

Figure 2. The plasmid map of PYT313 donated by Dr. Yongtao Zhu indicating the presence of the sacB, erythromycin resistance (ermF), and the promoter of F. johnsoniae, ompA (Zhu et al., 2017). 

Four primers are designed to isolate the GldB (gliding motility) gene within the C. Lytica DNA and are then used to create a new suicide vector using PYT313. As shown in figure 3, primers a and d contain restriction enzyme sites on their 3’ and 5’ sites, respectively. These sites correspond to specified restriction enzyme sites on the PYT313 vector. Primers c and d are homologous 1 kb upstream and downstream of the GldB gene from the start and stop codons, respectively. Through three polymerase chain reactions (PCR), the AB fragment and CD fragment are used to create the AD fragment which contains the GldB gene with restriction enzyme sites upstream and downstream of the DNA (Francis et al.). 

 

Figure 3. Four primers are designed for Overlap PCR. Through two PCR rounds, the gene is removed from the bacteria C. Lytica and ligated onto the PYT313 suicide vector. Figure from (Francis et al.). 

Then, through double digestion, the PYT313 vector is digested at the two specified restriction enzyme sites. After running the gel purification through electrophoresis, the larger digested PYT313 DNA is extracted and ligated with the AD fragment containing the GldB gene. This creates a new vector specifically designed to replace the GldB gene within C. Lytica with an inactive copy of the gene through transformation and conjugation processes (Francis et al.). 

Transformation and Conjugation 

Bacterial transformation is the process of environmental DNA uptake by competent cells. In this project, chemically competent E. Coli S17 λ Pir cells are used to uptake the GldB gene-inclusive PYT313 suicide vector. S17 cells allow for better DNA transfer during conjugation, which is why DNA uptake during transformation is crucial for GldB gene deletion. (Chen et al.) Then, the transformed E. Coli S17 cells are conjugated with C. Lytica cells for a direct transfer of DNA. 

Bacterial conjugation directly transfers genetic material from the E. Coli S17 λ Pir cells to the C. Lytica. During the conjugation, the mutant GldB gene is introduced to the recipient C. Lytica. As shown in figure 4, a two-step homologous recombinant event occurs: first and second crossover. 

During the first crossover, C. Lytica acquires the plasmid from the S17 cells, including the ampicillin antibiotic resistance. The conjugated bacteria is isolated using antibiotics, and a second crossover event occurs using the SacB sucrose counter-selection gene. During this event, the remaining part of the vector is removed from the C. Lytica, including the ampicillin resistance, leaving behind the mutant GldB gene or a wild-type GldB gene. Colony PCR is then conducted to differentiate between the two outcomes. C. Lytica cells with mutant GldB gene are grown and tested for results (Old Reliable: Two-Step Allelic Exchange by Bitesize Bio). 

Figure 4. Two Step Homologous Recombinant Event occurs, creating two types of bacteria. First conjugated C. Lytica has a wild-type allele, and second has the desired mutant allele. Adapted from Old Reliable: Two-Step Allelic Exchange by Bitesize Bio. 

Projected Outcome 

To test if the combination of DNA transfer is successful, the conjugated C. Lytica cells are introduced to antibiotic ampicillin. If the cells survive, the conjugation was successful, otherwise the cells would deteriorate. Additionally, successful conjugation will result in the C. Lytica cells losing their ability of iridescence. 

Results 

Spring 2024 

During the Spring 2024 semester, getting results on the transformation and conjugation was emphasized rather than altering the PYT313 to create a new vector. C. Lytica and PYT313 were grown in BB2 Agar and LB Agar plates respectively, as shown in Figure 5. Afterwards, a 50 ml culture was made using the colonies from both plates. Additionally, a 50 ml culture of S17 λ pir cells was grown for transformation. Transformation protocol was conducted using the S17 cells and PYT313. Transforming the S17 λ pir cells using the PYT313 was successful as it resulted in colony growth on LB Agar plates. Colony growth on multiple plates with antibiotic present is shown in Figure 6.

Figure 5. Growth of C. Lytica and PYT313 in BB2 Agar and LB Agar plates. LB Agar plate has ampicillin antibiotic added to it which demonstrates PYT313 ampicillin resistance. 

Figure 6. LB Agar plates with PYT313 suicide vector transformed S17 λ pir cells. Growth shows successful transformation due to the presence of antibiotic ampicillin. 

However, conjugation was unsuccessful, as no growth was present in the Conjugation Plates after a week of incubating. This could be due to multiple factors such as too many antibiotics or less cell density of C. Lytica or transformed S17 cells before conjugation. Additionally, due to time constraints, multiple trials of transformation and conjugation could not be completed. 

Summer 2024 

During Summer 2024, our aim is to establish which primers to use for the PYT313 for the two- step allelic exchange and complete successful transformation and conjugation with the new vector. Because we know transformation is possible, and conjugation can also be achieved with multiple trials, we hope to achieve complete deletion/replacement of the GldB by the end of summer. Additionally, the OUR grants have greatly enhanced this project by providing funds for resources and have made my research goals possible.

References 

ChenInês, et al. “The Ins and Outs of DNA Transfer in Bacteria.” Science, vol. 310, no. 5753, 2 Dec. 2005, pp. 1456–1460, www.ncbi.nlm.nih.gov/pmc/articles/PMC3919525/, https://doi.org/10.1126/science.1114021. 

DeSimone, Mark, Development of Genetic Engineering Tools for the Iridescent Bacteria Cellulophaga lytica, A Thesis (2021) 

 Francis, Matthew S, et al. “Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components.” Methods in Molecular Biology, 12 Nov. 2016, pp. 11–31, pubmed.ncbi.nlm.nih.gov/27837478/, https://doi.org/10.1007/978-1-4939-6649-3_2. 

Accessed 13 June 2024. 

Hall-Stoodley, L., Costerton, J. W., & Stoodley, P. (2004). Nature Reviews Microbiology, 2(2), 95–108. https://doi.org/10.1038/nrmicro821 

Kientz, B. et al. A unique self-organization of bacterial sub-communities creates iridescence in Cellulophaga lytica colony biofilms. Sci. Rep. 6, 19906; doi: 10.1038/srep19906 (2016). 

McBride, M. J., & Zhu, Y. (2013). Gliding Motility and Por Secretion System Genes Are Widespread among Members of the Phylum Bacteroidetes. Journal of Bacteriology, 195(2), 270–278. https://doi.org/10.1128/jb.01962-12 

“Old Reliable: Two-Step Allelic Exchange.” Bitesize Bio, 17 July 2018, bitesizebio.com/41461/old-reliable-two-step-allelic- exchange/#:~:text=The%20idea%20behind%20suicide%20vectors,understandably%2C%20can’t %20replicate. 

Pati, A., Abt, B., Teshima, H., Nolan, M., Lapidus, A., Lucas, S., Hammon, N., Deshpande, S., Cheng, J.-F., Tapia, R., Han, C., Goodwin, L., Pitluck, S., Liolios, K., Pagani, I., Mavromatis, K., Ovchinikova, G., Chen, A., Palaniappan, K., & Land, M. (2011). Complete genome sequence of Cellulophaga lytica type strain (LIM-21T). Standards in Genomic Sciences, 4(2), 221–232. https://doi.org/10.4056/sigs.1774329

Unabia, C., Hadfield, M. Role of bacteria in larval settlement and metamorphosis of the polychaete Hydroides elegans. Marine Biology 133, 55–64 (1999). https://doi.org/10.1007/s002270050442

Examining the Effect of Vitamin D on Melanoma

 By Mary Goodrow

 

Introduction 

Over the summer and during the spring semester, I was introduced to research and working with live animals. I also learned to keep a lab notebook and record lab activities during this introductory process. The first thing I did was separate fertilized fish eggs from unfertilized ones. First, it was hard to distinguish fertilized eggs from unfertilized ones. However, it was cool to see under the microscope. Later, after the semester had ended, I started learning how to make agarose plates. Agarose plates are needed in the process of producing Zebrafish with melanoma, which is done via recombinant DNA technology. It was a fun learning process being introduced to the lab equipment and procedures for using it. One of the graduate students was nice enough to offer some help along the way. I made some mistakes, especially since I am not used to the sterile technique. I made the mistake of placing the lids of the plates on the counter. It’s now ingrained in me to open the top of the lid only slightly when working with Petri dishes.  

Methods 

In my lab notebook, I kept track of all the instructions given to me by Dr. Ferreira, mainly the ones directly related to the Agarose plates. Each page was formatted based on the date and name of the procedure we performed that day. I initially listed the procedure for my main experiment, following space set aside for a table of contents.  

Results 

Despite the premature discontinuation of the experiment, I am proud to share that my agar plates were a success. Though new to me, the process of changing the tanks and feeding the Zebrafish was also carried out successfully. This first-time experience, without any prior knowledge, was a testament to the effectiveness of our approach, even in the face of unexpected circumstances. 

Discussion/Turn of Events 

In a significant turn of events, I have made the difficult decision to continue my academic journey at Vanderbilt University as a Biomedical Engineering student. This decision was not easily made, and it brings with it the uncertainty of being able to complete my experiment before my departure. However, I am certain that the lessons I have learned will undoubtedly shape my future endeavors.  

Recreating a Recombinant R.opacus Bacteria that Can Use Chitin

By Jackie Ramirez

 

Introduction

In 2022, roughly 119 million pounds of American lobster (Homarus americanus) were landed, and this catch was valued at around $515 million. With this gigantic haul of seafood, consumers will eat <50% of the animal, which makes up the lobster meat. The majority of lobster biomass is inedible and is discarded by homes, restaurants and other facilities, and the majority of that waste is lobster shell. The lobster shell contains three main constituents: minerals like calcium carbonate (CaCO3), proteins and chitin/chitosan polysaccharide. Of these shell components, chitin and chitosan have shown value in bio-based processes. Chitin and chitosan are carbohydrate polymers consisting of the amino sugars N-acetyl-D-glucosamine (glcNAc) and/or D-glucosamine (glcN) monomer units. Depending on the degree of acetylation of the polysaccharide, the polymer may be called chitin or chitosan, where the majority of the monomer concentration of chitosan is D-glucosamine. Chitin and chitosan are very attractive biomaterials with a range of household and industrial uses. Regardless, there remains a large percentage of lobster shells that are discarded or underutilized.

Chitin as a biomaterial for biofuel production is a promising and new area of research that will contribute to solving the global climate crisis. Chitinase ChiA, ChiB, and ChiC break down chitin into monomers of N-acetyl glucosamine (NAG). ChiA is an endochitinase that breaks down chitin within a polymer. ChiB and ChiC are exo-chitinases that cleave monomers at the end of a polymer. The monomers produced by these enzymes are used to produce triacylglycerols (TAG). From here, the triacyclglycerols can be trans-esterified into biodiesel. The bioengineering department here at UMass Dartmouth has looked to the surrounding South Coast of Massachusetts as a source of chitin for biofuel production. The shells of crustaceans comprise of 40% chitin by weight. Through research efforts at UMass Dartmouth, chitin has been derived and separated from the protein components of lobster shells (1). This ecofriendly extraction method has given researchers here the ability to utilize crustacean waste from human consumption to isolate chitin and use it for biofuels in conjunction with Rhodococcus Opacus (R. Opacus). R. Opacus, strain PD630, is a gram-positive microbe which will accumulate TAG in the presence of a steady carbon source. It’s high lipid storage ability and rapid turnover rate make it an excellent candidate for biofuel production (2). Chitin is a proposed carbon source for the bacterium. R. Opacus, which is unable to produce the chitinases necessary to break down chitin into its monomer counterparts for biofuel production, therefore this project will make a recombinant strain of R. Opacus to express and secrete chitinase enzymes.

Soon after receiving an OUR grant, my mentor and her collaborators changed the strategy to use the shells. R.opacus is a difficult bacteria to genetically manipulate, so they decided to get another bacteria that is easier to manipulate and has a better chance of taking up plasmids that have the chitinase genes on them. We switched to pseudomonas aeruginosa.

Methods

Genomic DNA isolation using the Promega gDNA isolation kit.

Design primers specific for ChiA from the bacteria S. marcescens and amplify the gene from the genomic DNA.

Initial PCR conditions: Using 5ul gDNA, 1ul of ChiA-For primer and 1ul ChiA-rev primer plus Taq Supermix. The reaction proceeded with standard PCR cycle parameters with annealing at 59 degrees Celsius and 45 cycles.

Second Attempt using a temperature gradient to see what temperature is ideal for primers to anneal to the template.

Figure 1: We tested 57 degrees upto 62 degrees. Each bar indicates the temperature in that well.

Third attempt PCR: We switched to using Q5 high-fidelity Taq DNA polymerase.

Figure 2: These are the PCR Parameters we used with High Fidelity Taq.

Results

Initial attempts to amplify the ChiA, ChiB and ChiC genes from s.marcescens gDNA (Figure 3). The faint bands at the bottom of each lane are primer dimers. We are expecting bands between 1.0kb and 1.5kb. After some research we decided to try using a Taq polymerase that had High Fidelity. The reason was because we are trying to find one gene in a genome of 5,241,455 bp, and we figured that a DNA polymerase that could stay associated with the template better might allow us to get the genes. The High fidelity Q5 DNA polymerase resulted in the expected products between 1kb and 1.5 kb (Figure 4).

                        

Figures 3 (L) and 4 (R): First attempts to amplify ChiA, B and C. 

Having figured out how to get the correct bands I focused on Chitinase B. I was able to amplify ChiB and gel purify only the correct sized band (Figure 5).

Figure 5: Gel purified ChiB genes

The project is being continued by another student. The next steps are to cut the ChiB insert with enzymes and insert it into the vector.