Engineering Success

Design

Last year we utilized a green fluorescent tag to check for successful insertion of our plasmid, this was unhelpful due to the green color of C. reinhardtii so we replaced it with an mCherry tag so that the fluorescence was red, which would be easier to distinguish from auto-fluorescence.

Build

We inserted the mCherry tag into 4 different strains of C. reinhardtii through electroporation. The mCherry tag was inserted at the C-terminus of our nosZ gene so that if it was expressed it would show expression nitrous oxide reductase.

Test

We tested for mCherry expression by Western blot and immunofluorescence. Initial test are inconclusive and will be repeated with new controls.

Learn

A major problem faced while doing the testing is the ability for C. reinhardtii to auto-fluoresce which makes it difficult for us to distinguish from expression of mCherry or just typical cell function. Next year we will utilize a different tag to test for successful expression of our plasmid.

Design

Psr1 is a transcription factor that initiates transcription and expression of phosphate intake channel protein. Normally, this gene is only expressed by C. reinhardtii under conditions of phosphate starvation. We theorized that by adding a copy of the gene under the Psad promoter, it will always express regardless of the level of phosphate in the cell, increasing phosphate uptake from the environment.

Build

Built the pL1 Psr1-nanoLuc plasmid using the C. reinhardtii MoClo cloning kit and Psr1 genes from the Alison Baker Lab using golden gate cloning.

Test

Confirmed proper construction of PL1 PSR NanoLuc using a restriction enzyme digest on a 1% agarose gel, and verified through sequencing.

Learn

Plasmid construction occurred correctly in E. Coli and can be used in future builds to test phosphorus uptake and luciferase expression.

Design

Knowing that pL1 Psr1-nanoLuc was properly constructed, a Level 2 plasmid was constructed using aadA to combine spectinomycin resistance in order to test incorporation and expression in C. reinhardtii.

Build

We completed the L2 plasmid using golden gate cloning methods.

Test

Proper plasmid construction was confirmed using restriction digest on 1% agarose gel and then further confirmed via sequencing. Properly constructed pL2 aadA Psr1-nanoLuc was used to transform C. reinhardtii using electroporation. C. reinhardtii was then allowed to grow before testing. We performed a phosphate uptake assay, comparing the rate of uptake of our transformed chlamy compared to untransformed control.

Design

We designed a plasmid to express nosZ, Ptc amiRNA and Psr1 simultaneoiuisely.

Build

We assembled the plasmid and confirmed that it was assembled correctly using restriction digest.

Test

We electroporated the plasmid into chlamy and screened nearly 200 colonies in order to find several that had luciferase activity. However, we found that chlamy transformed with this multi-gene plasmid grew more slowly than control chlamy, and had phosphate uptake levels similar to that of untransformed chlamy.

Learn

There may be limits to how much exogenous DNA chlamy can incorporate, and still express efficiently.