Team:Wageningen UR/Notebook/Niels
Automated Xylella fastidiosa detection tool
By: Niels Appelman
This is Niels his notebook. Here you can read about how Niels researched our detection device. The result of his work can be found on his result page.
May
Week 1 (13th of May – 19th of May)
Defined my subproject.
Week 2 (20th of May – 26th of May)
First met with dr. Saggiomo to brainstorm on the detection device. We had multiple subsequent meetings throughout the project. Defined my subproject.
Week 3 (27th of May – 2nd of June)
Defined my subproject.
June
Week 4 (3rd of June – 9th of June)
Started in the lab to familiarize myself with basic lab techniques (PCR, gel electrophoresis, Gibson assembly etc.) Further defined my subproject.
Week 5 (10th of June – 16th of June)
Continued labwork to familiarize myself with basic lab techniques. Wrote thesis proposal.
Week 6 (17th of June – 23rd of June)
Continued labwork to familiarize myself with basic lab techniques.
Week 7 (24th of June – 30th of June)
Continued labwork to familiarize myself with basic lab techniques. Defined questions that should be answered for the design of the detection device. Researched biology of insect vectors and how it would be applied in my project.
July
Week 8 (1st of July – 7th of July)
Continued labwork to familiarize myself with basic lab techniques. Familiarized myself with LAMP. Made a first proposal of LAMP experiments to perform. Researched biology of insect vectors and how it would be applied in my project. First came up with feeding assays.
Week 9 (8th of July – 14th of July)
Continued labwork to familiarize myself with basic lab techniques. Collected necessities to capture insects. Researched biology of insect vectors and how it would be applied in my project.
Week 10 (15th of July – 21st of July)
Started Labwork for my project. Made media to attract insects and amplified the RimM gene from the Xylella fastidiosa gDNA. Defined all entomological experiments I wanted to perform.
Week 11 (22nd of July – 28th of July)
Tested (Harper et al., 2010)’s LAMP primers on X. fastidiosa gDNA. Redefined all LAMP experiments I wanted to perform.
Week 12 (29th of July – 4th of August)
Attempted to determine the Limit of Detection of X. fastidiosa genomic DNA. Results were inconsistent. This may be attributed to the inherent difficulties in handling large pieces of DNA. First test into the interference of a sucrose medium on LAMP. Redid an experiment from a prior project to find LAMP primers for a cas9 DNA element.
August
Week 13 (5th of August – 11th of August)
Vacation.
Week 14 (12th of August – 18th of August)
Confirmed the proper functioning of cas9 LAMP primers. First determined heat of LAMP reaction was enough to thermolyse E. coli transformed with detectable plasmid. Pursued possibilities to perform entomological experiments in an entomology lab. Wrote protocols for performing entomological experiments.
Week 15 (19th of August – 25th of August)
Tested the interference of different concentrations of sucrose in LAMP detection of X. fastidiosa gDNA.
Week 16 (26th of August – 1st of September)
The first Philaenus spumarius (Spittlebug) membrane feeding trials were performed. Insects died fast. This was later attributed to a lack of humidity in containers used.
September
Week 17 (2nd of September – 8th of September)
More P. spumarius membrane feeding trials were performed. Insects died fast. This was later attributed to a lack of humidity in containers used. Got new containers for insect experiments.
Week 18 (9th of September – 15th of September)
First Golden Gate assembly of plasmid containing the X. fastidiosa DNA corresponding to the specificity of Harpers primers (part of the RimM gene). This failed as a one-pot reaction. First membrane feeding assay with Cicadella viridis (Green leafhopper).
Week 19 (16th of September – 22nd of September)
Golden Gate assembly was retried as a 2 step reaction (1. Restriction digestion 2. Ligation). E. coli DH5a was transformed with resulting plasmid, and positive result was verified with colony PCR.
Week 20 (23rd of September – 29th of September)
The created X. fastidiosa model bacterium was used to determine the Limit Of Detection of Harper’s primers on whole bacteria. Made competent cells. Explored Arduino and coding in Arduino. Made a plan for building detection device. First found C. viridis feeding on PCR tubes.
Week 21 (30th of September – 6th of October)
Explored bioinformatics to create better LAMP primers. Transformed cells for transmission assays. First performed transmission assay on C. viridis with 96 wells plate. This was unsuccessful due to contamination, but I did confirm the effectiveness of the head-dissection technique used to determine the presence of bacteria in insects. Started working on detection device prototype.
October
Week 22 (7th of October – 13th of October)
Performed paraffin encasing experiment. This was successful, as enzyme activity was present in the encased mastermix. Finished the circuitry of the detection device prototype. Performed second transmission assay on C viridis using PCR tubes. This was successful and transmission of bacteria was established.
Week 23 (14th of October – 20th of October)
Performed last C. viridis transmission assay.
References