Team:Imperial College London/Lab Diaries/XylE team
|Lab Diaries||Overview | Surface Protein Team | XylE Team | Vectors Team | Modelling Team|
|Here are the technical diaries for our project. We've split them up into three lab teams and the modelling team. We think it's really important that absolutely anyone can find out what we've been doing. For a really detailed look at what we did, and when, you've come to the right place!|
|Typical XylE assay|
we constructed the standard E.coli promoter J23101 with sticky ends. These ends are complementary to restriction sites made by EcoRI and SpeI enzyme. This promoter will be later used in 3A assembly to construct a promoter-RBS-XylE design in a psB1C3 vector. E.coli will be transformed with this final construct plasmid to assess XylE activity and characterization. It will also be one of the submitted BioBricks.
these cultures are going to be used tomorrow for mini prepping. Mini prep will allow us to isolate E.coli 's plasmid DNA(which contains the XylE gene).
Mini prep is usually used to confirm that our gene of interest has not been changed in any way, as the isolated plasmid id sent for sequencing. However, since XylE was taken from the registry, we assume that it is fine and no sequencing is required. The mini prep will later be used for the midi-prep (that gives out higher yields of DNA needed for cloning).
gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine the volume ratios of samples to be used for 3A assemply ligation
The J23101 gene in a BioBrick vector containg RFP gene
Performed gel analysis on the purified XylE and J23101 to obtain ratios for ligation. First gel was scrapped as it produced appalling (explanation for Nick: really bad) results, 2nd gel run was successful.
Tuesday 7th Sept
Wednesday, 8th Sep
Thursday, 9th Sep
Friday, 10 sep
The transformation was a SUCCESS. 2x replica plates were made plate 1# 1-6 plate #2 6-11; colony 6 and colony 9 of plates 1 and 2 respectively were transfered into a 5ml liquid culture + 5μl CmR. These will later be turned into glycerol stocks. After the replica plates have grown up mini preps on a number of colonies shall be performed - this hopefully will eliminate the contaminating plasmid DNA. This will be followed by a midi prep.
Saturday, 11 Sep
The transformation of E.Coli with PSB1-C3 with insert did not work :(
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
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
Weds 15th September
Thurs 16th September
- 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
Friday 17th September
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 :-(
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
Wed 22nd Sept
Thurs 23rd Sept First day of AUTUMN!!
Fri 24th Sept
Monday 27th Sept
Tues 28th Sept
Weds 29th Sept
Thurs 30th Sept
Friday 1st Oct
ahahahaha awesome. hey you :)
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!
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
Friday 8th Oct
Sunday 10th of the 10th of the 2010!!!!! and im in LAB
Monday 11th Oct
Tues 12th Oct
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|