Experiments
This page contains the notebooks that were kept for each wet-lab experiment, in addition to the induction week and interlab study protocols. For more details on these experiments, including detailed explanations as to their rationale, refer to the wet-lab section below.
Wet Lab Experiment Links
Standard Protocols
Throughout the experiments, our team used a set of standard protcols. This enabled us to produce batches of reagents in advance and troubleshoot our epxeriments to a greater degree.
Click the link for the standard protocols
Quantification of Auxin biosynthesis in E.Coli
The auxin expression experiment aims to prove our bacteria are able to produce auxin for algae growth after taking up the plasmid with the 3 auxin genes (Aro8, Ald-H, and KDC). The first part of the experiment includes inserting all g-blocks (Aro8, Ald-H, and KDC) into the vector backbone (pBbE8c-RFP) through golden gate assembly followed by transformation. The second part of the experiment aims to show the auxin expression through measuring auxin concentration by UPLC.
Click the link for the Auxin procedures
Light SwitchCre-LoxP recombination Experiment
The Light Switch experiment aims to prove our bacteria are able to change their genetic expression from one set of genes to another, via induction using a specific wavelength of light. The first part of the experiment involved cloning the LoxP gblock into a vector backbone, before transforming into bacteria. The second part of the experiments then involved testing the fluorescence of the MKATE2 before inducing a genetic change by transfecting with the Cre Plasmid. Transformed and induced cells were then tested for the fluorescence expression of MKO2, which comprised the final section of the experiments.
Click the link for the Light Switch procedures
PhoU knockdown for phosphate accumulation
The phosphate-accumulation experiments aims to show that our bacteria are capable of taking up phosphate from a growth medium and sequestering it in their cells. We seek to starve the microalgae of phosphate to trigger lipid accumulation. The firs part of the experimental procedure is concerned with the clonning or our pdCas9 sysytem and the transformation of our competent E.Coli. The second section explains the use of our vanadate molybdate colorimetric assay to detect phosphate sequestration.
RFP silencing via introduction of PdCas9 plasmid and light induction
The RFP silencing experiment aims to validate our genetic switch mechanism through the activation of LoxP, leading to the knockout of Red Fluorescent Protein encoded in the genome of the bacteria. This will provide a simple visual cue as to whether the genetic rearrangement leads to a change in expression. The first part of the experiment involved introducing the RFP pdCas9 plasmid into a strain of RFP genome encoded E.Coli. This was followed by induction the plasmid via Cre and IPTG. the second section of experiments involved identifying the loss of fluorescence of the RFP caused by the CRISPR knockout.