Team:UPF Barcelona/Proof Of Concept

Team:UPF Barcelona - 2021.igem.org

Wetware

Wetware Proof of Concept

Experiment conditions and details.

Introduction

Here we present the studies and results regarding the proof of concept of our project’s wetware part. The aim is to establish the feasibility and rationale key points that support ARIA’s further research development and use in more advanced stages. Due to this, all experiments have been conducted under controlled conditions to respect procedural consistency and thus achieve the highest reproducibility of the results.

Given that at a bureaucratic and legal level it is very difficult to work with human samples, and the resolution of requests takes a long time, we have used synthetic constructs. These consisted of isolated plasmids containing dsDNA templates for resistant genes, that is, the gRNA target from our biosensors.

The gRNAs have been modified and optimized during the project. The final version consists of the spacer sequence both followed and preceded by a direct repeat (DR), which enhances gRNA transcription and target DNA recognition.

Biosensors that can successfully detect antibiotic resistance sequences

Tests with biosensors based on CRISPR-LbCas12a technology are supposed to result in a fluorescent signal measured in RFU if the designed gRNAs (see Design) identify the antibiotic resistance bacterial target sequence. If this is accomplished, the biosensors are proven to work.

The conditions that have led to a successful detection are detailed below in Table 1 and correspond to measures accomplished using a biosensor with LbCas12a (BBa_K2927005) and the Efficient gRNA Chloramphenicol construct (BBa_K3791021) in the “joint approach” and the addition of an isolated plasmid containing a dsDNA template for the Chloramphenicol resistant gene.

Replicates	3 biological replicates
Induction	[IPTG] = 100 µM
Lysis	Lysis type	Enzymatic lysosome lysis: 10 mM Tris HCl pH 7.5 100 mM NaCl
Lysis	EDTA	without EDTA
Detection	Reagents	DNaseAlert Bulk Substrate: 5 µL 10x DNaseAlert Buffer: 5 µL Cell lysate: 36 µL Target sample: 4 µL (plasmid containing resistance gene to Chloramphenicol: 90 ng/µL) Total volume: 50 µL
	Approach	In vivo - in vitro “joint approach” (lysate from the same cell culture, cotransformation)
	Temperature	37ºC
	Kinetic cycle	2h with reading intervals of 5 min

Table 1: Optimal conditions for the detection of antibiotic resistance genetic sequences using CRISPR-Cas12a.

The fluorescence results for BBa_K2927005 + BBa_K3791021 (Cas12a + gRNA for Chloramphenicol resistance gene) in Figure 1 range between 26031 and 21607 RFU and are coherent since the one with sample needs to be higher due to the gRNA - sample sequence match. The decrease in fluorescence signal is only 16.99% over 2 -hour time, which indicates a maintained stability of the signal.

The biological significative negative control (without sample) produces a signal which is 38.1% lower in RFU than the one given by the biosensor with its corresponding sample. It ranges between 16118 and 14461 RFU. This is attributed to the endogenous nuclease activity present in the cell lysates. In both cases, the fluorescence is much higher than in the blank (negative control with water), a fact that is consistent.

Therefore, the results confirm our engineered biological system serves as a biosensor and accomplish the purpose for which it was created.

This figure shows a graphic of the fluorescence measurements for gRNA for Chloramphenicol resistant gene+Cas12a, a negative control (without sample) and a blank.

Figure 1: Fluorescence measurements for gRNA for Chloramphenicol resistant gene+Cas12a.