Protocols

Core Molecular Biology Protocols

: This method is used for assembling new plasmids by joining multiple DNA fragments together. It is performed following main 3 steps:
I - PCR amplification to linearize the vectors and generate the overhangs needed for insertion.
II - PCR clean up to get rid of all the PCR residues.
III . Cloning Assembly to assemble the different fragments together and obtain a circular plasmid

: The goal of preparing competent cells is to make bacteria able to efficiently uptake foreign DNA (like a plasmid) through transformation. In our case, we prepared them for electroporation..

: Electroporation is one of the methods used for transformation, alongside chemical transformation (which involves calcium chloride and heat shock). In this method, a high-voltage electric pulse is applied to create temporary pores in the cell membrane, allowing plasmid DNA to enter.

: This method allows to check the length of the DNA in our cells to confirm whether the plasmid had been fully inserted.

: Gel extraction is used when PCR produces multiple bands, allowing us to isolate only the correct fragment. However, this method is slower and provides a much lower yield than the standard PCR clean-up protocol.

: Sequencing is performed to determine the exact nucleotide sequence of the amplified DNA fragment and confirm if the targeted sequence is present and correct.

: This method is used to grow bacteria in a nutrient-rich liquid medium, allowing them to multiply and reach a high density for further experiments.

: This method is used to preserve bacterial strains for long-term storage by mixing them with glycerol, a conservation agent.

: Small-scale procedure to isolate, extract and purify plasmid DNA from a bacterium. Usually made as a stock for further experiment like transformation of a new strain.

: Method for making nutrient agar plate in petri-dish for bacterial growth.

: Streaking bacteria is a method used to isolate single colonies from a glycerol stock on an agar plate, which can then be used to start a liquid culture from a single colony.

: If received dry, primers and synthetic DNA need to be resuspended for further utilisation.

: During our project, multiple solutions of various chemicals were made. Here is a general protcole to prepare aqueous solutions.

Rare Earth Elements Protocols

: This protocol aims to assess how much REE our binding proteins can capture by incubating them with engineering curli-producing E. coli and quantifying both unbound and protein-bound REE using the Arsenazo assay.

: We aimed to prepare fully dissolved REE chloride solutions by dissolving REE oxides in 0.5 M HCl, then diluting to workable 1 mM stock solutions in MilliQ water.

: This method allows us to recover binding proteins produced by our engineered bacteria, which are too large to be secreted and therefore require cell disruption for extraction.

: The Congo Red microplate assay is a quantitative method used to measure bacterial amyloid production, especially curli fibers.

All the visual figure templates created for the protocols have been published in the BioRender library to be shared with fellow biology researchers.

Experiments

Protocols

Core Molecular Biology Protocols

Rare Earth Elements Protocols

Primers Table

Experiments

Protocols

Core Molecular Biology Protocols

Rare Earth Elements Protocols

Primers Table

Project

Name

Purpose

Target

Sequence 5'- 3'

Size (bp)

Temperature (°C)

Paired with