Team:Queens Canada/Notebook/Week17

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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.  
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.  
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<div id="September" class="contenttitle">  
<div id="September" class="contenttitle">  

Revision as of 03:39, 27 October 2012

Control

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  
05/09/2012PCRPhillipmgfp-5  
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

August

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.

September
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.

October
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.