Team:LUBBOCK TTU/Results


· Ligated fragments coding for sfGFP and mNeonGreen into YEp352 vectors
· Roughly confirmed presence of fragment through agarose gel
· Performed transformation of S. cerevisiae BY4741 with plasmids roughly confirmed to contain our biobricks

— Digestion/Ligation of YEp352 vector and Reporter Gene Fragments —

· Digested YEp352 vectors were ligated with either our sfGFP biobrick or our mNeon Green biobrick after PCR cleanup to remove one of the enzymes that was not heat killed
· Samples of sfGFP ligated vectors were ran in parallel to mNeon Green ligated vectors
· The ligation product was transformed into E. Coli for cloning
· Cells were mini-prepped to extract our plasmid
· To confirm the extracted sample consisted on plasmid containing our fragment of interest, a gel was run
· To prepare for the gel check, a portion of the mini-prep product was digested
· The enzyme should cleave the plasmid on either side of the previously ligated fragment, resulting in two pieces of DNA, the cut vector (approx. 5kb) and the naked fragment (approx. 2 kb)
· Figure 2 shows a clear line at 5kb and faint bands at 2kb, indicating that the fragments should be contained with mini-prepped plasmid

Fig. 1. 1kb DNA ladder
from NEB

Fig. 2. Agarose gel confirming presence of fragment within vectors.

— Next Steps —

Fig. 2. Experimental setup. This image shows how [Ca2+] has varied among wells
of the plate. This concentration setup was consistent among all 3 plates.

Using the 96 well plates, with Ca2+concentration gradients as shown above, we will incubate in a range of temperatures and measure the fluorescence output
· Given this data, we can investigate a more focused range of calcium concentration

— Considerations for replicating the experiment —

· We used common wet lab procedures requiring common wet lab equipment.
· All protocols are listed under the Experiment tab and provide clear instructions for those interested in replicating our work.