Rare Teso · Biotech

01.

Bacterial Leaf Scorch and Xylella Fastidiosa

2025

Genspace

Tools: CRISPR + CAS9

Bacterial Leaf Scorch (BLS) is a plant disease caused by the bacterium Xylella fastidiosa. It poses a growing threat to vegetation by forming harmful biofilms that block water and mineral pathways and resist plant immune defenses. BLS impacts plants, trees, and crops, causing reduced agricultural yields, declines in urban canopy health, and big-time economic harms for farmers.

BLS is spreading alongside the emergence of agricultural monocultures and loss of genetic diversity among plants. The experiment my labmates and I did explored how bioengineering tools can offer targeted and sustainable solutions to plant diseases.

.

When biological foundations are disrupted, people lose the psychic structures that help them make sense of reality.

Petri Colonies

02.

Objectives

Our main tools were CRISPR-Cas9 technology. We used this biotech combination to target two genes involved in the fastidian gum biosynthesis pathway: gumD (the initiating enzyme) and gumH (the fastidian gum elongation enzyme α-1,3-mannosyltransferase). Our goal was to design a synthetic DNA fragement containing a modified plasmid with a functional CRISPR-Cas9 complex to be inserted into Xylella fastidiosa cells, inducing double-stranded breaks in the target Xylella genes. In this multistaged process, we focused on inserting DNA guide sequences into a plasmid with the CRISPR-Cas9 machinery. Understanding the X. fastidiosa lifecycle enables the successful targeting of specific enzymes and control this hardy, plant-damaging bacterium.

02-1

Putting in wetlab work

02-2

Pentasaccharide repeat comprising glucose, mannose, and glucuronic acid

02-3

Gum operon and fastidian gum synthesis

03.

Methods

Our approach was simple but difficult due to a small cutting area.

Step 1 — Plasmid Preparation

Plasmid Preparation & Transformation: We amplified pCas9 plasmids in E. coli DH5α under chloramphenicol selection. Competent cells were transformed via heat shock and cultured overnight at 37°C.

03-1

Plasmid map of E. coli DH5α

03-2

Heat-shock competency & overnight culturing

Culture Growth & Plasmid We established overnight starter cultures in LB media with chloramphenicol. Large-scale cultures (250 mL) were grown to OD 2-3, and high-quality plasmid DNA was isolated using GeneJET Maxiprep Kit with endotoxin removal.

Step 2 — Culture Growth & Isolation

03-4

Maxiprep with endotoxin removal columns

03-4

Maxiprep with endotoxin removal columns

03-5

OD 2–3 culture before plasmid extraction

Step 3 — Guide RNA Design & Cloning

Guide RNA Design & Cloning: We designed guide RNAs to target gumD and gumH genes (essential for biofilm formation). Oligos were phosphorylated, annealed, and ligated into Bsal-digested Cas9 plasmids using T4 ligase.

03-6

Targeted Direct Repeat Sequences

03-9

Mathing TAE buffer volumes

03-7

T4 ligation into BsaI-digested pCas9 backbone

Step 4 — Gel Verification

Verification: We analyzed digested plasmids by running an agarose gel electrophoresis (2% gel, 100V, 20-30 min) with ethidium bromide staining. We excised and purified target bands using Monarch DNA Gel Extraction Kit.

Running 2% agarose gel to confirm insert ligation

03-8

Purified Samples Excised for Gel Electrophoresis

03-9

Running 2% agarose gel to confirm insert ligation

Step 5 — Final Transformation

Final Transformation: We transformed ligated pCas9-gRNA constructs into competent E. coli DHSa for amplification and downstream applications targeting Xylella fastidiosa biofilm disruption.

Transforming the ligated plasmid and plating colonies

03-11

Post-ligation transformation on LB + Chlor

03-12

Ambiguous colony — possible contamination

03-13

Final transformation outcome

04.

Results

The DNA concentration was too low for reading on both E-gel and sequencing.

+pCas9 colonies were successfully propagated, demonstrating that our control was run without contamination. Chlor-resistant plasmids grew in the presence of Chlor. The protocol yielded a small but visible colony. It is uncertain whether this colony was a successful transformation or a contaminated mold growth.

The samples never reached the necessary optical density for a successful maxiprep. The first overnight culture was overgrown, which may have caused a rejection of the pCas plasmid from early in the experiment. A separate overnight culture was refrigerated, stopping growth.

Ligation was not successful. The two-fold hypothesis for this is:

1. oligo design that led to almerization

2. possible problems during the annealing step.

According to the Maraffini protocol, plasmid overhangs were four base pairs, creating potential stability issues for the formation of a working ligation product.

04-1

Fig. 1 — Control plate streaking (negative transformation)

04-2

Fig. 2 — Successful population growth

04-3

Fig. 3 — Potential Contamination

04-4

Fig. 4 — Running an Egel

04-5

Fig. 5 — Failed sequencing due to insufficient DNA

05.

Discussion

Synthetic biology is a unique tool for investigating life cycles of plant pathogens. Our protocol demonstrated plasmid engineering and colony growth utilizing a well-established CRISPR platform. We focused on plasmid modification and operon insertion to target transport and synthesis of xantham via gum operons. This experimental process builds on highly customizable CRISPR technology with artificially created DNA oligo sequences. We modularly added to isolation and purification literature with choice Maxiprep techniques.

Iterative informed E-gel electrophoresis tests allowed us to improve our experimental transformation. Future protocols may be optimized in a variety of ways. One useful technique would distinguish between the absence v.s. low quantity colony yield of pCas9 plasmid DNA production. Future experiments may optimize ligase and oligo selection in addition to agar plating quantity. PCR testing is one of many possible analysis routes that can clarify results gathered via E-Gel and sequencing.

The potential bacterium uptake of engineered plasmids opens up ethical questions on the altering of Xylella fastidiosa. This engineering protocol may introduce unwanted resistance and gain of function changing improving pathogenicity, as well as expanding the list of potential hosts. However, the need to study and control plant pathogens keeping usage of pesticides/antimicrobial chemicals as low as possible is a preffered research outcome.

04-6

plate growth verification

06.

References

All photo credit due to Maren Fossi

Simpson, A., Reinach, F., Arruda, P. et al. The genome sequence of the plant pathogen Xylella fastidiosa. Nature 406, 151–157 (2000). https://doi.org/10.1038/35018003

Sambrook J, Russell D, Sambrook J. The Condensed Protocols From Molecular Cloning. 1st ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2006.

Guan W, Shao J, Davis RE, Zhao T, Huang Q. Genome Sequence of a Xylella fastidiosa Strain Causing Sycamore Leaf Scorch Disease in Virginia. Genome Announc. 2014 Aug 21;2(4):e00773-14. doi: 10.1128/genomeA.00773-14. PMID: 25146135; PMCID: PMC4153481.

Biology and Integrated Management of Xylella fastidiosa subsp. pauca (Xanthomonadales: Xanthomonadaceae) on the olive tree, Olea europaea L. (Lamiales: Oleaceae) - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Graphical-representation-of-transmission-of-X-fastidiosa-to-olive-trees-Almeida-2016_fig2_351614681 [accessed 27 Jul 2025]