Part:BBa_K4654011

Li+-II_Riboswitch

The riboswitch is the lithium-sensitive Li⁺-II riboswitch which was described by White et. al(2021)¹. They created the sequence by constructing a consensus sequence from several riboswitches of a lithium sensitive riboswitch class¹. It is a translational riboswitch that forms a three-dimensional structure on the mRNA in the absence of lithium ions, inhibiting translation by masking the ribosome binding site in the spacer sequence 2. Once lithium binds to the structure, a part of it unfolds so that the RBS becomes accessible to the ribosome which leads to translation of the downstream gene.

Figure 1: This figure shows the schematic work mechanism of the riboswitch-reporter-construct.

Usage and Biology

For the detection of lithium, we utilize this riboswitch in our system to generate a signal in the presence of Li⁺. A riboswitch is an RNA construct with the ability to exert control over gene expression, either positively or negatively, upon binding to a specific ligand. These molecular devices operate at either the transcriptional or translational levels within the cell by forming two domains: An aptamer and a ligand binding domain. The aptamer domain catalyzes the folding into a three-dimensional structure, thereby forming the ligand binding domain. The Li⁺-II riboswitch is a translational riboswitch. Translational riboswitches are encoded on the DNA (Figure 1(A)). Upon transcription, they form an aptamer structure in the 5‘-UTR of mRNA, that masks the ribosome binding site. As a result the ribosome cannot bind to the mRNA and the mRNA is not translated (Figure 1 (B)). Once a ligand binds to the aptamer structure of the riboswitch, its structure changes, revealing the ribosome binding site and translation can proceed (Figure 1(C)). Accordingly, we harness the Li⁺-II riboswitch to activate the expression of our reporter system in response to lithium. The signal allows us to quantitatively determine the lithium level in the samples.

White et. al (2021) described two riboswitches: Li ⁺ -I and Li+-II. Since Li+-I exibits strong resemblance to a sodium binding riboswitch and binds sodium ions with only a few mutations in the sequence, we decided to only work with the Li+-II riboswitch¹. In bacteria, the lithium riboswitches are are often associated with nhaA genes. NhaA proteins are membrane-localized Na⁺/H⁺ antiporters that also transport Li⁺, thereby enabling the bacteria to eject toxic Li⁺ from the cell.

Results

We verified the correct folding of the riboswitch in the absence of lithium and the unfolding once lithium is present by cloning a reporter gene downstream of BBa_K4654011 (BBa_K4654017, BBa_K4654018, BBa_K4654019, BBa_K4654020) and cloning this construct into E. coli bacteria carrying a gene encoding the T7 RNA polymerase. We then did liquid culture assays with or without the addition of lithium chloride. We also used cell-free systems to characterize the riboswitch outside the cell.

Figure 2: Construct 1G in Cell-Free Assay.Response of Riboswitch-sfGFP Construct 1G (BBa_K4654017) to 50 mM LiCl compared to an uninduced sample as negative control in a cell-free environment. The figure shows RFU values, the measurement was done with Tecan Spark Plate reader, the settings for the plate reader are in the protocol "sfGFP / mScarlet-I3 Assay". For cell-free protein expression we used the biotechrabbit RTS E. coli HY KIT. The experiments were conducted according to the manufacturer's instructions. 50mM LiCl was added to induce sfGFP expression. The result graph shows clearly that there is no reporter gene expression in the absence of lithium ions but reporter expression once lithium is present. From this, we concluded that BBa_K4654017 includes the DNA sequence for a fully functional riboswitch that folds on mRNA level when lithium is not present but unfolds once lithium is added to the growth media. Sequence and Features BBa_K4654011 SequenceAndFeatures