Notebook

Introduction

In this section, you will find a detailed record of our daily work in the laboratory. Every experiment we carried out is described here, following the protocols presented in the protocols section.

To make it easier to navigate, we have organized the notebook into two parts. First, you will find an overview that summarizes all the experimental work at a glance. Then, we provide a more detailed document where each day is explained step by step.

Both parts are connected through a color-coding system, which allows you to quickly match the overview with the corresponding detailed entries. This way, you can choose whether to get a general picture of our progress or to dive into the full details of our experiments.

Description of objectives and procedures

The overall goal of the project is to express four protein variants: Protein A and Protein B, both in their normal and extended forms. The extended form refers to the protein and its cryptic peptide. Subsequently, two DNA aptamers capable of specifically recognizing the variants containing the cryptic peptide will be designed and selected. Thus, we will obtain one aptamer for each extended protein.

PROTEIN CLONING AND EXPRESSION

Express and extract the proteins of interest. To this end, specific plasmids containing the genes of interest corresponding to Protein A / Protein A + PC / Protein B / Protein B + PC have been designed, along with the His-tag and GST-tag sequences necessary for subsequent purification.

The steps to be followed are as follows:

• Vector digestion: The plasmid TrcHis B will be digested using the restriction enzyme NheI
• Recombination and transformation
• Culture and miniprep
• Verification of positive colonies

PROTEIN PURIFICATION

According to the literature reviewed on expression conditions in E. coli (Bolton, S.C et al, 2020), the optimal growth temperature for Protein A is 16 ºC overnight, and for Protein B, it is 20-25 ºC overnight.

Two purification conditions will be carried out: one for each purification tag separately (His and GST), if needed a third one will be performed combining both (double purification). To achieve this, the protein purification protocol developed by Pep Clotet's group (Purification Protocol) will be followed. Harper and Speicher (2011) and Smith and Johnson (1988), demonstrate the GST purification method.

The objective is to establish the optimal conditions for obtaining the proteins of interest in Escherichia coli BL21 or RIPL.

The bacteria will be transformed with the corresponding plasmids and cultured. Protein expression will be assessed by Western blotting using antibodies against their tags, which will allow confirming their presence and estimating their production level. Based on the results obtained in the expression phase, a decision will be made as to whether to perform purification with a single tag or both.

Procedure:

• Miniprep transformation
• Colonies selection
• Grow until it reaches an optical density equal to OD 0.3
• Induction with IPTG
• Purification protocol
• Assess protein purity by Coomassie and/or Western Blot

APTAMER PREPARATION

A protocol from the Stockholm team (2021) will be used for aptamer selection. The process is based on the SELEX technique, which allows for the selection of DNA sequences with high affinity for a target molecule.

In this case, a DNA library (commercially available from IDT) will be used and incubated with the previously purified proteins of interest. After three rounds of SELEX, the sequences will be sequenced to identify and narrow down the candidate sequences with the highest affinity.

Once the most promising sequence has been identified, it will be modified using in silico design tools (Dry Lab) to optimize it for detection in a future diagnostic test.

This part of the work is expected to be performed solely computationally.

We rely on Zhao et al. 2020 and Aljohani et al. 2022, two papers that demonstrate the use of aptamers in diagnostics and the method of their production.

Overview

Notebook