Team:Queens Canada/Notebook/Week17

In August, our focus was mainly on creating our first working GFP construct. We managed to replace the variable D3 domain of fliC with GFP by PCR overlap extension and ligate is under the J04500 RBS and promoter. The day we saw fluorescent colonies on our plates was definitely one of the best days of the summer. Unfortunately, we did not have access to a scanning electron microscope to be able to visualize GFP on our flagella. After combing through some literature, we developed two different protocols for isolation of flagella: one involving shearing the flagella with a Waring blendor, and the other involving removal of flagella from cells by heat. We tested a culture of nFF GFP (no linker full fliC) + J04500 in pSB1AK3 as well as a culture of just J04500 in pSB1AK3 as a control, and managed to get fluorescent flagellar pellets in just our GFP construct. However, due to the possibility that there might still be cell debris contaminating the flagellar pellet, we ran SDS-PAGE on some protein samples. There was a distinct band at 70 kDa (the size of our protein of interest, a GFP-flagellin chimera) in our test sample that was not present in our control sample. Unfortunately, after all this characterization work, we discovered that our GFP construct had an illegal cutsite in it.

We also worked on PCR overlap extending metal binding proteins such as SmtA and fMT into the variable D3 domain of fliC, but were unsuccessful.

Our lab work continued into the fall semester, where we were hoping to build our ideal construct of a super chimera of five different proteins: flagellin, combined with a cohesin domain, bound a by a dockerin-enzyme-secretion tag combo. If it works, that’s five different proteins, each with a different function, from three different organisms. After realizing the illegal cutsite of GFP, we switched our focus to RFP. We managed to insert cohesin into the variable D3 domain of fliC through PCR overlap extension, as clone secretion tag+RFP+dockerin. Next came the task of ligating this construct under the B0034 (RBS) and J04500 (RBS+promoter).

Other parts that we PCR overlap extended parts into the D3 variable domain included: Rv2579 (dehalogenase), LinB (dehalogenase), RFP, GFP, xylE, and mgfp-5 (mussel foot binding protein for adhesion). We also managed to do a ligation of RFP replacing the D3 domain (our RFP deletion construct).

We converted some of our parts into the BB-2 standard using PCR (such as the two constant domains of fliC on either side of the variable D3 domain), but were unsuccessful in ligating them into the submission plasmid. Other things we worked on included xylE characterization using catechol, and motility assays using various strains of E. coli expressing various parts.

We obtained fliC deletion strains from the Yale Coli Genetic Stock Centre (CGSC): JW1908-1 and YK4516. We made these strains competent by the CaCl2 method. Then we successfully transformed our constructs into JW1908-1, grew liquid cultures, and performed motility assays. After approximately 18 hours, no motility was shown in the flagellin, J04450, RFP deletion, or RFP insertion constructs. Our future work will include performing motility assays for the rest of our constructs, transforming our constructs into YK4516, and sending out parts out for sequencing. It appears that we have successfully ligated s-doc-RFP under the J04500 promoter, evidenced by the appearance of a few tiny fluorescent colonies on a ligation plate, so we are continuing to work on our super chimera of 5 different proteins. We are planning to perform experiments with our sdoc-RFP cultures to determine whether the dockerin-RFP proteins are successfully being secreted extracellularly, and if so, whether or not they can latch onto cohesin-expressing flagella.