Week 11
Monday 13th september
Overnights weren't set up on sunday so they were made up alongside some assay cultures. J23101-XylE-B0014 colonies 8 and 10 of the replica plate #2 were picked. Chris also provided a replica plate containing 3k3 vector colonies. This was over a year old and he was unsure whether it was the correct plasmid or if the cells would grow up. I picked all available colonies 102 150 151 and 260 of kanamycin resistance. Lastly for assays 2x LB 2xM9 cultures were made 5ml +5ul antibiotic.
Tues 14th September
test confirming that yellow product of XylE enzmymatic reaction is leaks back from the cytosol into the solution.1C and 2C is the XylE producing cells after centrifuging and redissolving of the pellet and 1S and 2S is the supernatant after centrifuging
- assay on plate reader of:
- 0-0.75 initial O.D. of transformed E.coli
- 0-1mM initial catechol concentration
The assay was carried out with E.coli, top ten spcies, transformed with J23101-XylE-B0014 in pSB1C3 vector.The overnight culture wastransfered in new medium this morning for 4 hrs before assaying. LB medium was used for dilutions and blank. Catechol was diluted in ddH2O.
Data analysis of the assay
- Mini preps of XylE 8,10 colonies and 3K3;102 151 260 colonies. Analytical digests were performed using E+S on XylE, E+S AccI and HindIII+E for 3K3 vector respectively. The band patterns correctly identified the vector to be 3k3. Colony 10 of XylE and Colony 260 of the 3k3 vector were set up direct into 100ml LB and so will require til 12pm for midi prepping on weds. Nick performed a second plate reader assay to determine the minimal concentration of Catechol to use for assays. I took 990ul M9 culture containing colony 10 and 10ul catechol was added. This was incubated for 10 mins and then spun down. The supernatant was removed into a cuvette and the cells resuspended in M9 salts. The ODs were then read on the spectrophotometer at 380 and 600nm. It was found that control cuvette of M9 salts had a miniscule reading. M9 + cells ~0.007. Supernatant + cells were ~ 2.5 therefore we could deduce that the coloured catechol breakdown product is exported out of the cells.
Piotr:
- Mini prep of 1 sample (6 samples lost due to mistake) of GFP-Xyle fusion protein and its digestion with Spe and Xba (gel to be run on the next day)
- Preparing E.Coli colonies for the next day for mini prep to be redone
- PCR of 6 samples of GFP-Xyle fusion
Weds 15th September
- Midi preps of overnights.
- In preparation for assays determining the effect of catechol and/or breakdown product on cell viability we prepared Top10 cells, one strain containing pVeg-XylE-terminator and the other containing a CMR plasmid of similar size. As the XylE cells have already been prepared, only Top10 transformation with CMR-plasmid had to be carried out.
Thurs 16th September
- Top10 CMR transformation was successful. Four overnight cultures of each Top10 XylE and Top10 CMR were set off with either M9 or LB medium respectively.
- Piotr: Did mini preps from bacteria with blunt ended Xyle-GFP fusion and did diagnostic using both:
- digest with XbaI and SpeI
- PCR reactions with the following primers added: HIS-GFP, XylE-Xba and the other sample with HIS-GFP and GFP-flag
The PCR reactions were not very conclusive on the gels but digests allowed to determine that colonies 1->5 seem to have the right sizes of DNA in them and on Friday they will be prepared to be sent off for sequencing
- continuation of the midi from overnight step. A gel analysis was run which showed the correct bands were present. A gel purification of the protein was then performed and the bands excised.
Friday 17th September
- Catechol assay of transformed E.coli Top ten(J23101-XylE-terminator) in M9 medium + data analysis
The gel purifications of 3k3 vector and XylE were used for ligation.
3K3 was dephosphorylated and then ligated in a ratio 5:1 with the insert. This was left overnight and Chris tried to transform with it on sunday.. ligation and transformation failed :-(
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Week 12
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Monday 20th Sept
Performed ligation again. Errors in the sequencing we received back regarding J23101-XylE-B0014.
One was a log error (in XylE) phew. The other is in PSB1C3 out of the scar site and should be OK.
Chris is currently assembling the pVEG promoter+ RBS in a vector. Once he has finished this, I will combine XylE-GFP and XylE with this promoter for comparison characterization with J23101 (in E.coli and Bacillus)
Tues 21st Sept
- Ligation to be transformed in a bit. (Issue with the vector not being cut efficiently=failed ligations?)
- I am cutting the original XylE midi as well as PSB1C3 containing B0014 and ligating them today (hopefully) the vector is purified, waiting on the gel run and then extraction of XylE insert. A ligation ratio must be determined from a gel analysis then dephosphorylation of the vector and then ligation. This will produce a promoter-less XylE+terminator vector which will be readily available to switch in the desired promoter (E+S cut)
- Florian is performing a mini prep on pVeg promoter which if it is correctly identified in the gel i can use to ligate to my XylE + PSB1C3-B0014.
- Piotr is PCRing the reverse version of XylE that will be under the control of inducible promoter LacI (not delivered from synthesis yet) that will become a testing construct.
Wed 22nd Sept
- Piotr's PCR has been successful for 3 samples (1. recommended annealing temp; 2. recommended + 2 degrees and 3. recommended + 4 degrees). Sample no1. has been run on the gel and gel purified. It has been cut overnight with SpeI and tommorrow in the morning XbaI will be added.
- I (Maddie!) performed steps towards the ligation (XylE-3K3) again from scratch (inefficient cutting steps??) including digest, gel analysis and extraction.
- I ligated PSBC3-B0014 and XylE
- I picked successfully transformed colonies for a replica plate and set up overnight mini cultures of 1,2,3 ComC DE promoter FWD and 4,5,6 ComC promoter Rev
Thurs 23rd Sept First day of AUTUMN!!
- I determined the ligation ratio for XylE and 3k3 (1:3) performed dephosphorylation of vector then ligation, to be transformed along with the other ligation tomorrow hopefully.
- I did mini preps of the Comc DE promoters, performed a ES test digest and am currently waiting for gel results. I shall then set up overnight midis.
- Miniprep results were fabulous, Ive chosen colony 1 and colony 4 which are ComCDE promoters FWD and REV respectively. They are made up into overnight midi cultures for tomorrow.
Fri 24th Sept
- Midi preps of ComC DE promoters FWD and REV (colonies 1,4)
- to be followed by a digest. XbaI Pst for FWD (not known for the reverse currently) to insert XylE after it.
- Ligation with XylE and PSB1C3-B0014 possibly.
- Transformation of XylE-PSB1C3-B0014 and of XylEe-3k3 into E.coli laterz
- Hi Nick, hope revision is going well. Love from Team XylE xox
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Week 13
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Monday 27th Sept
- Successfull transformations: now have XylE-PSB1C3-B0014 (no promoter) and 3K3-J23101-XylE-B0014. No background plate colonies..many colonies for 3K3-XylE and a few for PSB1C3-XylE. Colonies have been replica plated (plus catechol assay plate for 3K3-XylE) and set up for mini prepping tomorrow.
- Sumo tagged XylE will be given to us by Kirsten at some point this week and we will have to purify it. This can then be used for in vitro testing and comparisons made with the linker-XylE protein.
- pVeg and ComC DE promoter FWD can be added into the promoter-less construct after it has been midi-prepped and digested accordingly. ComC DE promoter REV can be added onto the reverse XylE construct (PCR'd by Piotr) AFTER it has been put into PSB1C3 and is more manageable. Kirsten is taking care of the GFP-XylE fusion for now, including putting the pVeg promoter plus RBS infront of it (proving difficult.) We still need to receive LacI promoter to create the inducible test version of the fusion XylE-GFP protein.
Tues 28th Sept
- Performed mini preps and then E+S / E+S AND AseI digests of colonies 1-7 3k3-XylE and 1-3 C3-B0014-XylE respectively. Gel runs showed them to be the correct thing :)))))) so, 3k3-XylE final vector is now left as 2-7 minis (catechol assay test showed colony 1 was background so DO NOT USE) in my DNA box (orange tubes). Colony 1 of the C3-B0014-XylE was selected for midi prepping and will be performed tomorrow.
Weds 29th Sept
- Midi prep of XylE-GFP fusion protein along with C3-B0014-XylE. The C3-B0014-XylE midi will then be used in 2x cloning digests (E+S) to insert pVeg and ComC DE FWD promoters. These will be obtained and also digested E+S to get out of their current vectors a gel analysis and purification will be run of all 3 in parallel, followed by a ligation ratio gel analysis, dephosphorylation of vector then ligation.
- Primers for J23101-XylE arrive today so they can be used to PCR out J23101-XylE (From my J23101-Xyl-E midi) with blunt ends and this will be ligated into the final Spec vector so that we can get some data with Bacillus.
- J23101-XylE-B0014 XylE-3K3 and PSB1C3-B0014-XylE were all sent off for sequencing
Thurs 30th Sept
- I gel purified ComCDE FWD promoter, C3-XylE-B0014, pVeg vector and XylE-B0014. Did a gel analysis to get ligation ratio.
- Set up midi culture of J23101-XylE-B0014 colony 8
- Set up midi culture to repeat GFP-XylE midi prep
- Set up 6x minis (plus replica plate) of final Spec colonies 1-6
- PCR reaction using newly arrived primers to get blunt ended J23101-XylE.
Friday 1st Oct
- morning maddie, from earl's court! :)
ahahahaha awesome. hey you :)
- today I peformed ligations for ComCDE FWD promoter and PSB1C3-XylE-B0014 and also PSB1C3-pVeg and XylE-B0014 they will be left overnight and transformed by chris tmro thaaanks.
- I ran the PCR Kirill performed yday to get out blunt ended J23101-XylE on a gel; there was no template DNA! so I've just set up the reaction again... hopefully i'll purify it later for ligation into the final Spec vector that kyasha has maaaaade :))) then we can test XylE in Bacillus.
- I'm currently performing Midi's of my J23101-XylE-B0014 (running low) and of GFP-XylE (3rd time!)
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have a look :)
it's from John Hoppkins wiki 2008. There are one or two good ones!
Love: Before I heard the doctors tell The dangers of a kiss;
I had considered kissing you. The nearest thing to bliss.
But now I know biology and sit and sigh and moan;
six million mad bacteria and I thought we were alone!
Biology is the science of moving tiny droplets of invisible liquids from one tube to another.
The human body was designed by a civil engineer. Who else would run a toxic waste pipeline through a recreational area?
I have a hunch that the unknown sequences of DNA will decode into copyright notices and patent protections.
It has recently been discovered that research causes cancer in rats.
A mouse is an animal that, if killed in sufficiently many and creative ways, will generate a PhD.
Week 14
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Monday 4th Oct
Did a replica plate and catechol assay plate/ minis 2 6 7 9 12 16. (Successful Pveg-XylE transformation)
Tues 5th Oct
4 16 colonies were background the rest turned yellow. Mini prepped 2 6 7 9 12 16. Made overnight assay cultures of 3k3-XylE and C3-xylE
Weds 6th Oct
- Digested mini preps 2 6 7 9 12 16 with E+S will run on gel to determine if it's the correct plasmid, then will select a colony to midi prep tomorrow.
- Will perform the same assays already performed by Nick with the 3k3-XylE constructs to compare results.
- Just realised Im working with background colony 16!! disregard this after the gel run!
- I'm currently keeping my undigested PVeg-XylE minis in florians DNA box (orange lids and labels)
Thursday 7th Oct
- Labpartner, goodmorning and from greece!! :p
- Niiiiiiiiiiiiiiick!!! you left the country! you were too ugly. good luck babe!
- Midi prepped PVEG-XylE-B0014 in PSB1C3; will digest out the insert (E+S) and put it into 3K3(E+X). Then both the midi (in PSB1C3) and the ligation 3k3-PVEG-XylE-B0014 can be transformed into TOP10. J23101-XylE-B0014 in 3K3 Vector also needs to be put into TOP10 currently only the PSB1C3 version is in TOP10. We need the rest for comparison testing as TOP10 is the more widely used testing strain.
- Florian kindly digested PVeg-XylE-B0014-C3 with E+S
- Ran PCR to get out blunt PVeg-XylE-B0014 using primers. Did 3 reactions at variations around 60 degrees.
Friday 8th Oct
- Ran gel analysis of my PCRs 1, 2 and 3* appear to have worked. Samples 1 and 3* show stronger bands and so should be used in the next ligation step into the SPEC vector (which kyasha will be doing) then transformation into Bacillus (meeee.)
- Chris is purifying digested PVeg-XylE-B0014-C3 to get the insert out and this will then be ran on a gel alongside cut (E+X) 3k3 to determine a ratio for ligation.
- Meeting today at 4pm, no one is here!! it'll be me florian ben and piotr attending..yikes
Sunday 10th of the 10th of the 2010!!!!! and im in LAB
- I did ligations of PVeg-XylE into 3k3 and of Reverse-XylE into the digested C-Tev LacI vector.
- I also set up assay cultures for monday 2x PVegXE 2x XE-C3 2x XE-3K3 and 2x GFP-XylE!!!
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Week 15
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Monday 11th Oct
- No Nicolas Kylilis, what a douche!
- Diluted my overnights 250ul into 5ml to grow up to around 0.5 OD
- Gonna perform assays with them this afternoon after some growing time :)))))))
- Doing data analysis with my last assay results comparing high copy (1C3) vs low copy (3K3)and the effects on XylE activity..boring
- Finalising the polo-shirt logo design, it's looking fiiiine, we're gonna look goooood ;)
Tues 12th Oct
- Transformations worked! we now have PVEG-XylE in 3k3 and reverse-xylE with lacI promoter (C-TEV vector)i did replica plates, catechol assay plates and overnights for minis. Will mini prep tmro and transform them into TOP10 and we should be good to go with all constructs.
- Assays messed up, my fault :( 3k3 didnt respond to catechol, so i think that I picked a background colony, i wont pick it again. The results are a bit ridiculous and will probably be chucked.
Weds 13th Oct
mini prepped 3k3-Pveg-XylE-B0014 and went to the school workshop! set off overnight midi of culture 9
Thurs 14th Oct
Midi prepped 3k3-Pveg
Fri 15th
Transformed all my constructs into T0P10 3k3-J23101/pveg and PSB1C3-J23101/PVEG
Output Photo Gallery
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