Team:Queens Canada/Notebook/Week17

Through the learning phase we researched intensively on the full flagella construct and what was required to incorporate protein infusions. As stated before, the D3 domain in the flagella construct has no real function, and can therefore be replaced with a protein of our choice. Through PCR overlap extension we would incorporate GFP and RFP into our constructs. To prove that it PCR overlap extension would worked, we would incorporate this PCR product containing the full fliC construct with no linkers with the GFP replacing the D3 domain (this was labelled nFFGFP - no linker Full Flic GFP construct) into the T7 promotor J04500 vector that contained the chloramphenicol resistance through digestion/ligation processes. We grew these ligations overnight and expected colonies.

As expected there were several colonies on each plate, to prove that the ligations worked only the colonies with green fluorescence were used for liquid colonies. Unfortunately we did not have access to an electron microscope, therefore we could not visually see the proteins on the flagella. Researching various flagellar isolation protocols , we developed two tested protocols to isolate the flagella containing GFP, which can be found in the protocols section. We also had a batch of J04500 controlled bacteria made to act as the control that would not contain protein infusions.

Essentially these two protocols would shear the flagella off of the cells using blender and heat techniques. The cell debris would be removed by centrifuge, and the sheared flagella would be collected as pellets. These isolated pellets when observed under the fluorescent microscope did glow green. Since there is a possibility that some cell debris would be present in the flagella pellets, we decided to run SDS-PAGE to see if there was a different between the controlled bacteria that would only contain flagella but not protein infusions. What we predicted was that at there should be a protein band at 70 kDa, which would be due to the GFP protein infused on the full fliC construct which would not be found on the control bacteria. Through running a few SDS- pages and comparing the control vs. fluorescent flagella bacteria, there was a distinct darker band at 70 kDa that was not found in the control. This means that it is likely that the flagella pellets in our nFFGFP (no linker Full Flic GFP construct) contained GFP proteins, which the J04500 bacteria did not. However due to an illegal cutsite, we changed our focus to the RFP characterization and the cohesin dockerin.

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.

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.

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.