Notebook

Central accomplishments(s)

Overview and Preliminary Plans for Peptide Production and Binding Affinity Testing

Wet Lab

Today’s goal was to establish a comprehensive plan for producing a peptide with binding affinity to perfluorooctanoic acid (PFOA) using E. coli as the expression system. We outlined the following steps: preparation of the cell line, plasmid production for peptide transfection, and the use of polyhistidine tags for purification. Following the purification using His-tag column chromatography, we will explore methods for His-tag removal and verify its effectiveness. Quantitative analysis using a DC protein assay will determine peptide concentration and impurity levels. We will then conduct binding affinity tests using ELISA, with control groups, including cheaper molecules like palmitic acid. Biolayer interferometry will be used to measure the dissociation constant (Kd) for the peptide-PFOA interaction. The protocol remains subject to optimization as the experiment progresses.

Central accomplishments(s)

Plan for His-Tag Removal and Transcytosis Experimentation

Wet Lab

Today, we presented our experimental plans to the Sode Lab, focusing on the removal of the His-tag from the purified peptide using TEV protease, with reference to the NEB protocol. We selected the E. coli strain BL21 for peptide expression. An additional experiment was planned to investigate the transcytosis of the engineered peptide, following procedures from relevant mouse studies. We also considered direct peptide synthesis, with a final decision pending based on time and cost assessments.

Central accomplishments(s)

Plan for Transformation of E. coli BL21

Wet Lab

Our focus was on the transformation of E. coli BL21 with the plasmid containing the target peptide sequence. We decided to follow the detailed transformation protocol from Edvotek to ensure efficient transfection. The wet lab will proceed with this phase upon final review of plasmid preparations. In response to a request from Dr. Sode, the wet lab team was restructured. The new core members of the lab were identified as Grant Holland, Zaid Syed, and Nishant Dengi, with Sam Carew serving as an alternate. Other members are invited to shadow during research activities but will not actively participate in the wet lab moving forward.

Central accomplishments(s)

Determining Amounts of HSA and PFOA for Control Group

Wet Lab

The primary task for today was determining the required amounts of human serum albumin (HSA) and PFOA for control group experiments, referencing an article on HSA-PFOA interaction from ACS Publications. We also reviewed relevant data on albumin pocket Kd values to inform subsequent testing phases.

Central accomplishments(s)

Control Experiment Protocol and Team Meeting Notes

Wet Lab

The meeting today focused on finalizing the protocol for control experiments involving PFOA immobilization on a microtiter plate, using a human albumin ELISA kit. We discussed using PBS/MgCl₂ as a buffer and considered various surface material properties to optimize the binding of PFAS and albumin. Following immobilization, we plan to block non-specific binding sites and proceed with chemiluminescence measurements to generate a Kd curve for the control group. Testing will involve a range of albumin concentrations, with constant concentrations of PFOA and anti-albumin. The control group experiments will begin once we receive the recombinant protein vectors.

Central accomplishments(s)

E. coli Transformation and DNA Production

Wet Lab

We are officially beginning to work in the Sode Lab. Today's efforts focused on attempting to produce more DNA using DH5α bacteria; however, the attempt was unsuccessful. This will necessitate ordering more DNA soon. The E. coli transformation procedure was initiated but yielded no positive results. A detailed review of each step has been noted to troubleshoot this issue, as colonies failed to form.

Central accomplishments(s)

AI Test Culture and IPTG Test Culture

Wet Lab

Both the 106Fa4 and 84Fa4 samples did not colonize as expected. In response, we began smaller-scale test cultures, with 5 mL AI solutions and IPTG cultures for protein expression assays. The tests involved adjusting media volumes and concentrations, but the smaller-scale tests showed discrepancies in optical density (OD). The AI culture met the required OD (0.4), while the IPTG culture remained suboptimal at 0.04. This outcome suggests a need for increasing cell density in future experiments to improve reproducibility.

Central accomplishments(s)

AI and IPTG Test Culture Results and Cell Lysis

Wet Lab

Today's results showed that only the AI solution achieved the target OD, while IPTG still fell short. Post-culture, we centrifuged the samples and added BugBuster to initiate cell lysis, followed by urea treatment for the insoluble fraction. The process successfully separated soluble and insoluble proteins, which will be analyzed using serial dilutions. The samples are now stored at 7°C for overnight analysis.

Central accomplishments(s)

Protein Quantification Challenges

Wet Lab

Our glycerol-stored pre-culture samples appear to have degraded, leading to an unsuccessful protein quantification attempt. Despite this, the assay data yielded an R² value of 0.98, indicating strong linearity in the standard curve. The data were normalized, and further dilutions were prepared for the next round of testing. We standardized protein quantification results by calculating the lowest adjusted concentration for all samples.

Central accomplishments(s)

E. coli Transformation Setbacks

Wet Lab

The E. coli transformation procedure was repeated, but once again, no colonies developed. Agar plates were remade, and the issue is under review.

Central accomplishments(s)

Continued E. coli Transformation Setbacks

Wet Lab

Another E. coli transformation was performed. Plates were stored in a 4°C fridge with parafilm applied to prevent contamination. Several questions have been raised about the differences between proteins and their mutations, which will be discussed with Josh later this week.

Central accomplishments(s)

Adjusted Preparation for E. coli Transformations

Wet Lab

A fresh LB medium batch was prepared, and cultures were sterilized via autoclave in preparation for the next round of experiments. We have slightly adjusted the E. coli transformation procedure based on previous results, increasing the heat shock time to 1 minute and switching from LB to SOC medium to improve transformation efficiency.

Central accomplishments(s)

Miscommunication about DNA Fragments vs. DNA Plasmids

Wet Lab

A miscommunication with the dry lab was identified; instead of plasmids, DNA fragments were being inserted into E. coli cells, leading to failures in bacterial growth. This issue explains why none of the bacterial colonies survived on the kanamycin plates, as the bacteria lacked the resistance gene. PCR testing is planned to confirm no contaminants are present.

Central accomplishments(s)

Restriction Enzyme Digestion and Results

Wet Lab

The first restriction enzyme digestion was performed on five vials. Each vial contained 1 µL of DNA and was digested using NC01 and HinD3. A 1% agarose gel electrophoresis was run, but visualization via UV light suggests more consistent dye incorporation is necessary for improved results.

Central accomplishments(s)

Ligation Challenges and Plasmid Formation

Wet Lab

The DNA ligation of PET-30a vector with the gene inserts was performed successfully. However, when transforming the DH5α cells, no colonies developed. It appears NC01 restriction enzyme was mistakenly used instead of Nde1, hindering the plasmid formation.

Central accomplishments(s)

DNA Quantification and Process Review

Wet Lab

No colonies developed again, prompting a DNA quantification test. Results indicated abnormally low concentrations, possibly due to incomplete ethanol washing during membrane preparation. We will repeat the restriction enzyme digestion and ligation, this time ensuring all reagent steps are performed correctly.

Central accomplishments(s)

Shift to Differential Scanning Fluorimetry

Wet Lab

A discussion with Sode lab team members highlighted that using perfluoroalkyl compounds for an ELISA/BLI assay is not feasible due to size constraints. A pilot study using DSF (Differential Scanning Fluorimetry) will be conducted once protein expression is complete. We are still investigating how the engineered albumin can bind and remove PFAS from contaminated environments. We are also considering potential applications of our protein in environmental biosensors or water treatments that remain promising. Further studies will explore these areas once the protein is purified.