Team:UC Davis/Notebook/Week 4


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Week 4

Due to difficulties in logistics, the DNA miniprep of BBa_K537003 and BBa_K537004 was not done over the weekend and the cultures had to be reinoculated in LB media. The DNA for BBa_K598001 was running low for further ligation experiments, so we decided to do another transformation with the use of dead stock. After we did a little bit of lab work, we met with Dr. Facciotti and Dr. Siegel to go over our construct and possible avenues of assembly. Dr. Siegel suggested varying the promoter and reducing the number of riboswitches that we would use for this experiment. He also suggested varying the copy number of the plasmids. By adjusting the promoter and copy number of the plasmid, possible sources of noise from TAL repression or unintentional riboswitch activation could be mitigated. With these considerations in mind, we decided to reduce the number of riboswitches that we would use for this construct and vary other aspects of our plasmids of interest.

We checked the transformation for BBa_K598001 and found colonies. We decided to inoculate one of the colonies in LB for further DNA amplification tomorrow. We also performed DNA minipreps on BBa_K537003 and BBa_K537004. The samples of DNA were then stored in the 4 degree room. We then discussed our construct in further detail about the use of possible promoters. We decided that the the use of inducible promoters would give us the greater amount of tunability, instead of trying constitutive promoters with fixed outputs. With our discussion from yesterday, we decided to recreate our list of oligos that we would order for synthesis. We also designed primers to fix the Bsa1 site in BBa_E0040 or BBa_E0240.

We discussed the idea of using inducible promoters in our construct with Dr. Facciotti and Dr. Siegel. Both agreed with the idea, since it offered the potential of additional tunability in our project and simplified the number of constructs that we would need to build. So instead of using two constitutive promoters, we have decided to use pTet and pBAD instead. We discussed several controls that would be necessary for our construct. We would need an arabinose promoter fused to GFP in order to ensure that pBAD was working as expected. In addition, we would need an extra positive control testing the function of our tetracycline promoter and riboswitch. Finally, we would need to use a control that would have a different TAL binding site that would not complement the TAL repressor to convey the specificity of our TAL repressors. After discussing our testing protocol, Dr. Facciotti suggested contacting Dr. Gallivan, an expert on theophylline riboswitches about any questions relevant to our project. In lieu of these discussions, we decided to hydrate BBa_K750000, a pBAD+GFP+Terminator construct. We then transformed the bacteria to amplify the amount of DNA and store in our glycerol stock for later use. We also performed a DNA miniprep on our cultures containing BBa_K598001. We plan on using this DNA for restriction digestion and ligation in order to do a test trial with an engineered riboswitch.

We also discussed using a graphical tool, KO3D, made by a former UC Davis iGEM student to help in modeling our data. KO3D is a program that allows one to look at a 3D plot from any particular angle. By measuring fluorescence and the concentration of different inducer molecules, one could deduce the behavior of their genetic circuit. We believe that using KO3D by other teams could also be a more effective means of part characterization and suggested this as a possible avenue of collaboration.

We spoke to Dr. Gallivan on the phone about his recommendations for possible theophylline riboswitches to use for this project and what concentrations of theophylline would be good for testing our construct. He suggested a couple of his lab’s past papers for us to look at for riboswitches. He also gave us input on whether different ligand-binding riboswitches could be made for the same general construct. The transformation of BBa_K750000 was partially successful with only one colony on the selective media. The colony was inoculated in LB broth for further propagation. Another restriction digest was carried out to reuse BBa_K598001 for another ligation reaction and standard assembly. After gel purification and ligation of our BBa_J23101+BBa_K598001 construct, we used the ligation mixture for a transformation with appropriate controls. We have also been continuing to refine our oligo designs for our constructs and hope to be able to order them tomorrow.

A small portion of the liquid LB culture containing BBa_K750000 was stored in glycerol. The rest of the rest of the culture underwent DNA extraction. We used a small amount of this DNA for site-directed mutagenesis. We intended to alter BBa_K750000, so that it may be compatible with Golden Gate Assembly and contain no BsaI sites. The attempted transformation involving BBa_J23101 and BBa_K598001 ligation mixture was unsuccessful. To deal with these difficulties, we decided to hydrate BBa_K598010, a composite part containing pBAD, a theophylline riboswitch, and GFP. We performed transformations for BBa_K598010 and pSB3K3, which could then be use for Golden Gate Assembly next week. While doing the pSB3K3 transformation, we realized that we did in fact have some DNA miniprep of pSB3K3 in our -20º C freezer. We decided to use this DNA miniprep for site-directed mutagenesis as well to remove BsaI. We would like to eventually use BBa_K598010 for a trial run involving our testing protocol. We finalized our primer list and hope to do Golden Gate Assembly with our proposed constructs next week.