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.

David and Faisal worked on making plasmids based on metal binding proteins, in the hopes of characterizing these parts with the flagella constructs. These parts included fmt, SmtA, and Pbr (lead binding protein). Some initial plasmids were successfully made through PCR, and PCR overlap extensions however due to some technical problems, we could not incorporate the metal binding proteins into the flagella construct.

Kevin and Andrew focused performed a lot of digestions and ligations for different parts, including Coh II, Dock I, RBS B0034, XyIE. They also tried to incorporate the flagellla constructs in different strains of E.coli to test for flagella motility.

Basically, the ideal construct that we're working for, is a super chimera of five different proteins. The flagellin, combined with a cohesin domain, which will be bound a by a dockerin-enzyme-secretion tag combo. If it works, that's five different proteins, each with a different function, from 3 different organisms.

Our lab work continued into the fall semester, where we are hoping to have our ideal construct. This construct that we were working for is a super chimera of five different proteins. The flagellin, combined with a cohesin domain, which will be 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 cut-site of GFP, we switched our focus on RFP. We tried to ligate S-doc RFP (secretion tag+RFP+dockerin) under the RBS B0034 and the RBS+ promoter J04500. Many ligations and digestions were doing during this month. This was a work in progress until October.

We also worked on making full constructs of flagella, by PCR overlap extending existing parts into the D3 variable domain, such as: cohesin, Rv2579 (dehalogenase), LinB (dehalogenase), RFP, GFP, and mgfp-5 (mussel foot binding protein for adhesion). We also managed to do a ligation of RFP replacing the D3 domain (our deletion construct).

We tried to convert our parts with the BB-2 standard using BB-2 primers, which we were successful in, however we attempted to ligate it into the submission plasmid, we were unsuccessful for these specific plasmids.

In October our s-doc-RFP constructs contain some fluorescent colonies, which we are currently testing with our cohesin flic expressing bacteria. We performed protein isolation of several parts which included; J04500, D3 fliC, D3 linB, RFP deletion fliC, D3 Rv2579 fliC, D3 RFP fliC, XyIE FC (deletion) fliC, and D3 Coh II fliC.

We got flic deletion strains from the Yale Coli Genetic Stock Centre (CGSC). These are e.coli strains that do not have native flagella. These strains include: JW1908-1 and YK4516. We made these strains competent by the CaCl2 method, to allow the cells to take up plasmid. 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. We are also 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.