Team:CSL Pittsburgh/Description

Our Project

Project Description

The honeybee, Apis mellifera, is an important pollinator that is suffering from a crisis called Colony Collapse Disorder (CCD). This phenomenon is a drastic decline in bee populations, disrupting wild ecosystems and global agriculture. Around the time we were picking our subject, we heard about CCD in the news and media and we were inspired to focus on this problem because it was a subject that affects our community. We recognized CCD as a local and global problem because bees are important to the wellness of the environment.

After further research, we found that CCD is caused by climate change, agriculture, pollution, and insecticides. Out of those four main causes, we decided to focus on insecticides, specifically the effects caused by imidacloprid. Imidacloprid is a neonicotinoid that interferes with the transmission of stimuli in the insect’s nervous system. Additionally, imidacloprid affects honeybees’ ability to forage for nectar and find their way back to the hive. If worker bees can’t bring back resources to the hive, it destabilizes the colony. Since we had the micro and molecular biology resources, as well as equipment, and help from professionals, we decided to take on this challenge. Of the four causes of CCD (climate change, agriculture, population, and insecticides), we thought that focusing on the effect of insecticide, specifically imidacloprid, was the best area to apply synthetic biology.

Through synthetic biology we inserted a gene found in Nilaparvata lugens (the brown planthopper) into a protein secretion system, allowing us to genetically engineer bacteria that can culture the bee. Our goal is for the honeybees to show similar resistance as Nilaparvata lugens to the insecticide imidacloprid. This will hopefully contribute to the stop of CCD, since the bee’s mobility and navigation will no longer be affected.

Methods

Our procedure was conducted in two general parts: molecular and clinical trials (using bees). The goal of the molecular protocols have been to genetically engineer bacteria to express the target functions. In order to do so, we used a number of molecular techniques to prepare the competent cells, transform those competent cells and plate on selective media, digest the plasmids with restriction enzymes, and ligate the genes of interest to create a new plasmid.

Figure 1.) Colony PCR of piGEM transformants.

PCR was performed on 3 colonies collected from the transformation of Part BBa_K112624 from the iGEM repository into DH5𝝰 E.coli using Phire green master mix with a 5 minute denaturation first step to lyse the cells and release DNA. VF2 and VR primers that anneal on either side of the inserted DNA show amplification of the part insertion for 1 of the colonies tested on our transformation plate. This plasmid was called piGEM for the remainder of the experiment for ease of identification.

Figure 2. Synthesized oligonucleotides ordered from IDT to be ligated into the pSB1C3 linearized backbone. The top oligo contains only the CYP6AY1 gene codon optimized from the brown plant hopper driven by the promoter and RBS from part BBa_S05355. The bottom oligo was designed in the same way with the addition of a pelB secretion signal and 6x His tag as an alternative method to complete the experiment in case of any issues with other plasmid parts.

Bee Trials

With the bee trials where we treat the bees, our goal was to treat the specimens with solutions consisting of the wildtype, pCYP, and pBEE transformed bacteria, respectively, and observe how the bees react to those treatments.

• Prepared 50% sucrose solution for feeding bees during laboratory testing.



• Grew overnight cultures of pCYP, pBee, and DH5α and measured the optical density of each culture using a spectrophotometer.



• Prepare 5mL food solutions for each test (pCYP, pBee, DH5α) with standardized bacterial concentrations (approx. 10% bacterial culture, 90% of the 50% sucrose solution).



• Gravity fed the corresponding solution to each trial bee enclosure using a trimmed 3mL transfer pipette (24 hours).



• Administering Imidacloprid Prepared 5mL of imidacloprid solution using 50% sucrose (calculated quantity of imidacloprid based on LD50 of 0.1079 uL/bee).



• Gathered data on number of dead and live bees in each enclosure (T= 0hr).



• Gravity feed solution to bees using a trimmed 3mL transfer pipette (1 hour) .



• Gathered data on number of dead and live bees in each enclosure (T=1hr).



• Replace imidacloprid solution with 50% sucrose solution.



• Gathered data on number of dead and live bees in each enclosure at T=24hrs and T=48hrs.



• Anesthetized bees by placing enclosure into -20°C freezer for 1 hr.



• Submerged anesthetized bees into soapy water solution

Results

Both the bacterial cultures and the bees were treated with imidacloprid. The results of these treatments were collected and analyzed using statistical methods.

Figure 3.) Normalized difference in optical density of E. coli cultures grown in varying concentrations of imidacloprid. Wild type and engineered E.coli were grown in various Millimolar concentrations of the pesticide imidacloprid for 24 hours. The optical density of the culture was recorded for 3 trials and averaged for each strain of E.coli. The data was normalized to the average OD600 of each bacteria grown without any imidacloprid added to the media. Error bars represent standard error of the mean. This shows that concentrations of 1mM, 0.1mM, 0.01mM, and 1µM of imidacloprid all produce statistically significant differences in growth with p-values of 0.001070978, 0.038314144, 0.00568822, and 0.022970616 respectively, when analyzed with a two tailed t-test.

Figure 4. Secreted His-tag protein expression from wild type and engineered E. coli cultures. Three cultures of wild type and engineered E.coli were grown overnight then reinoculated to grow to log phase. These cultures were diluted to 1.0 OD600 and 10 mL of each culture was spun down at low speed. The supernatant of each culture was run through His-pur Ni-NTA resin membrane to trap the 6xHis tagged proteins in the supernatant. The concentration of protein was then evaluated using the Nanodrop 2000C at an absorbance of 280nm and averaged across the 3 trials. A statistical difference was not observed across the test groups when evaluated with a student’s t-test. However a large difference was seen and this experiment may need optimized to ensure the His-tagged proteins are being captured properly. Standard deviations were 0.102825094 and 0.525094595 for the wild-type and engineered E. coli respectively.

Figure 5. Survival of Honey Bees exposed to imidacloprid when treated with different strains of E. coli. Wild type and engineered E.coli were grown to log phase and fed to honey bees in a sugar meal at 0.01 OD600 for 24 hours. Imidacloprid was then fed to the bees in an oral LD50 dose (0.107ug/bee). The survival of the bees was assessed at 24 and 48 hours. This shows that after 48 hours a statistically increased survival was seen when the bees were cultured with the EpBee E. coli expressing the imidacloprid resistance gene. Error bars represent standard error of the mean.When assessed with chi-squared analysis the p-value after 48 hours between the two test groups over 3 trials was 0.000247362