Team:Queens Canada/Notebook/Week17


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Notebook - Week 17 and onwards

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol
19/08/2012PCR overlap extensionAndrewxylE XS and PP eCFP XS into nFF GFP + J04500  
19/08/2012DigestionAndrewSmtA and Fmt with XS  
20/08/2012GelDavidDP fmt XS, DP SmtA XS, eCFP full construct PCR product  
20/08/2012PCR overlap extensionAndrew   
23/08/2012Liquid culturesKevinC. thermo x-dockerin II pet21b, CelD dock I pet1bb, C cell. Trx-dock pet32, C cell. cohesin pet32, C therm CipA Coh I pet21b, C therm CohII pQE30 , CipA Dockerin C therm pQE30,pCip7 (Coh7) C therm pQE30 ,CelD Dock C therm pQE30, C therm dock II pQE30  
24/08/2012MiniprepsKevinsee above  
27/08/2012Heat shock transformationPhillipLinB pDE5TI7  
30/08/2012Liquid culturesKevinnFF GFP J04500 in DH5a, XL1 Blue, TOP10  
9/1/2012PCR amplification of D3 domainKevin   
01/09/2012PCRKevinRv2579, Coh2, Doc2, xDoc2  
01/09/2012GelKevinxylE XS  
01/09/2012GelKevinD3, GE LinB, GE xylE XS  
03/09/2012PCR OEKevinLinB, CohII, Rv2579, D3  
03/09/2012Digestion with DpnIPhillipLinB, CohII, Rv2579, D3  
03/09/2012MiniprepKevinlinB, CohII, Rv2579, D3, DH5a + J04500  
05/09/2012PCR of RFPDavid   
05/09/2012Digestion with XPPhillipMPP LinB, rv2579, CohII, D3 full constructs, DH5a + J04500  
05/09/2012PCRPhillipsee above  
14/09/2012Digestion with EPKevinJ04500, RFP FC J04500, CohII FC, D3 FC, Rv2579 FC, LinB FC  
15/09/2012Liquid culture and miniprepKevinxylE FC  
15/09/2012GelKevinRFP, xylE FC, D GFP, D mgfp, D CohII, D xylE, D LinB, Rv2579  
15/09/2012LigationKevinD3 domain into psb1c3  
16/09/2012GelBeinimgfp, xdoc, doc, xylE FC EP GE, D RFP GE, D mgfp, D cohII, D linB, D Rv2579  
16/09/2012Digestion with SPBeiniMPP DH5a + J04500, RBS B0034 enzymatic purification using Biobasic kit
16/09/2012Liquid cultures and miniprepKevin, PhillipD3 FC Ligation trials  
18/09/2012LigationsKevinxylE FC GE and RFP FC GE into pSB1C3  
18/09/2012PCR overlap extensionPhillipD RFP, D GFP, D mgfp, D cohII, D linB, D Rv2579 (all GEs)  
18/09/2012Heat shock transformation of overlap extensions and ligationsKevinsee above  
19/09/2012Liquid culturesKevinlig RFP, D3 RFP OE, D3 GFP OE, D3 linB OE, D3 Rv2579, D3 mgfp OE, D3 cohII OE, D3 ctrl  
21/09/2012MiniprepsAaron, Beinisee above  
22/09/2012PCR OE of RFP with dockerin IVictorRFP, dockerin I7 trials with varying ratiosused sec tag left primer and dock right primer
22/09/2012Heat shock transformationBeinixylE FC AK into XL1 Blue  
23/09/2012GelFaisalxylE, cohII, linB, Rv2579, mgfp, D3 ctrl, sxdoc, sdoc  
23/09/2012PCRKevinsdoc and XS RFP doc with sec tag 2 primer  
23/09/2012DigestionsBeinisxdoc XP, xylE with SN, xylE with EP, CohII with SP, mgfp with EP  
23/09/2012GelPhillipsee above  
24/09/2012Heat shock transformationKevinJ04450, RFP D3 OE  
24/09/2012Glycerol stockKevinxylE full fliC deletion construct in pSB1C3 in XL1 Blue  
24/09/2012250mL cultureKevinxylE FC pSB1C3 XL1 Blue  
25/09/2012GelBeinigel extraction sxdoc RFP XP, sdoc RFP XP, xylE EP, MGFP D3 fliC EP  
25/09/2012Heat shock transformationBeiniMPP D3 FC lig into XL1 Blue, MPP D3 CohII OE into XL1 Blue  
25/09/2012LigationsKevinsdoc RFP J04500, sxdoc RFP J04500, mgfp pSB1C3  
26/09/2012Liquid culturesBeiniD3 FC, xylE FC AK, cohII FliC, mgfp5 FliC, sdoc RFP b0034, sxdoc RFP B0034  
26/09/2012MiniprepPhillipsee above  
29/09/2012Heat shock transformationsKevinD3 FliC, D3 LinB FliC, D3 Rv2579 FliC, D3 Coh FliC, D3 RFP FliC, RFP deletion FliC, J04450, mgfp5 FliC transformed into TOP10
30/09/2012Liquid culturesBeinisee above  
02/10/2012Protein isolationKevinmgfp5 FliC, J04450, D3 FliC, D3 LinB, RFP deletion FliC, D3 Rv2579 FliC, D3 RFP FliC, xylE deletion FliC, D3 CohII FliC5 mL cuturesheated 65C for 15 mins
02/10/2012xylE assayKevin   
03/10/2012Flagella isolation precedureKevin successful pellet in all samples


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.


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.