Revision as of 09:43, 6 September 2010

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<p id="popshow">Follow our progress: Click me!</p>
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 <ul id="poplist">
  <li><a href="#Week_6" class="popt">Week 6</a></li>
  <li><a href="#Week_7" class="popt">Week 7</a></li>
  <li><a href="#Week_8" class="popt">Week 8</a></li>
  <li><a href="#Week_9" class="popt">Week 9</a></li>
  <li><a href="#Lab_Diary" class="popt">Lab Diary</a></li>
 <li><a href="#Output_Photo_Gallery" class="popt">Output Photo Gallery</a></li>

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XylE team Lab Objectives

construction of XylE fusion protein
Testing expression of XylE in E.coli and characterization under the control of a constitutive promoter
Construction of the -ComE promoter/XylE fussion protein- expression system
Construction of the -LacI promoter/XylE fusion construct- expression system

Schedule

Week 6

Week 7

Week 7	Monday	Tuesday	Wednesday	Thursday	Friday
MORNING	midi-prep XylE E.coli (2hrs)	gel extraction kit on XylE gene trapped in agarose	3A assemply: make replica plates (overnight) Catechol assay of E.coli	Mini-prep XylE E.coli	Midi-prep XylE E.coli
lunch break
AFTERNOON	restriction digestion of XylE for 3A assemply gel purification of XylE from rstriction digestion	3A assemply of vector, XylE and J23101 promoter above construct transformed in E.coli	*primers arrived!	PCR extension of XylE and GFP, round 1

Lab Diary

Thursday, 12-Aug-2010

anealing DNA strands of J23101 promoter in a waterbath

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 assemply 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.

prepared two overnight cultures of XylE transormed E.coli (one 50microliters and one of 450 microliters)

these cultures are going to be used tomorrow for mini-prepping. Miniprep will allow us to isolate E.coli's plasmid DNA(which contains the XylE gene).

Friday, 13-Aug-2010

mini-prep of XylE transformed E.coli

Mini-prep is usually used to confirm that our gene of interest has not been changed in any way, as the isolated plasnid 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 yeilds of DNA needed for cloning).

gel analysis of plasmid DNA retreived from mini-prep of XylE transformed E.coli, cut with restriction enzymes. From light to the left, 50micrograms digested DNA : 50 undigested DNA : 450 digested DNA : 450 undigested DNA. In lanes 1 and 3 the smaller band has a size of about 1kB which corresponds to RBS-XylE gene. The bigger bands are the cut vectors. In lanes 2 and 4 is the uncut biobrick from the registry. It appears smaller on the gel than it actually is as circular DNA travels faster through the pores of agarose gel rather than linearised DNA.

Monday, 16-Aug-2010

midi-preped the XylE-transformed E.coli. The DNA yeild from the midi-prep was 134micrograms as determined by spectrophotometry. This is the XylE that is going to be used for all further experiments.
restriction digestion of midi-prepped XylE by Xbal and PstI to prepare it for 3A assemply. (with J23101 promoter and PSB1C3 vector)
gel analysis of the restriction digestion mixture to isolate XylE gene

I made an overnight culture of Bacillus

Tuesday, 17-Aug-2010

using the Gel Extraction Kit, we isolated the restriction enzyme cut XylE gene from the agarose gel lamp.
gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine their ratios for 3A assemply ligation
3A assemply of XylE, J23101 promoter and pSB1C3 vector.
Transformation of XL-Blue competent E.coli with the above construct.

gel analysis of XylE, J23101 promoter and pSB1C3 vector samples to determine the volume ratios of samples to be used for 3A assemply ligation

*I followed Chris's Bacillus transformation protocol to transform Bacillus with constitutive GFP and RFP DNA as well as a control without DNA

Thursday, 19-Aug-2010

We run the XylEGFP1 PCR reaction to construct the His-GFP-Flag and Linker-XylE-Spe construct.

Friday, 20-Aug-2010

The J23101 gene in a biobrick vector containg RFP gene

we run a gel on XylEGFP1 PCR reaction. Results: GFP was extended successfully, XylE extension FAILED (too much non-specific annealing)
catechol assay on 2hrs bench ligation of promoter,xylE and vector failed.
two replica plates of overnight ligated J23101, XylE and pSB1C3 transformed E.coli (one for catechol assay)
the His-GFP-Flag DNA was gel purified
transformation of XL-1Blue cells with J23101 in J62001 vector from the registry. One more attemp to construct a successful promoter-xylE ligation, since we believe that the strand annealed promoter was of bad quality

Week 8

Week 8	Monday	Tuesday	Wednesday	Thursday	Friday
MORNING	PCR extention of His-GFP-flag round 2	midi-prep registry obtained J23101 gel purification of XbaI-His-GFP-flag-TEVs	gel analysis of overnight ligation and gel separation gel analysis of linker-XylE-SpeI PCR reaction	make replica plate and catechol assay plate	catechol assay
lunch break
AFTERNOON	Gel purification of linker-XylE-Spe PCR construct Gel analysis of His-GFP-flag round 2 PCR rection and then gel separation of DNA	restriction digestion, gel analysis and gel purification of registry obtained J23101 PCR extension of linker-XylE-SpeI ligation of pSB1C3, J23101 and XylE (overnight)	gel purification of overnight ligation transformation of E.coli with overnight ligation product and selection by plating gel purification of TEVs-linker-XylE-SpeI construct	PCR construction of fusion protein	Gel analysis and gel purification of fusion Xyl E protein

Monday, 23-Aug

set up overnight cultures for midi-prep
gel separation of linker-XylE-Spe DNA FAILED
PCR reaction for extension of His-GFP-Flag
Catechol assay on E.coli transformed with overnight ligated J23101, XylE and pSB1C3. FAILED

Tuesday, 24th-Aug

midi-prepped registry obtained J23101. A yield of 130ng/ul of promoter was obtained. The promoter is in a biobrick vector called J62001. The promoter is upstream of RFP gene.
the vector carrying the promoter was digested with SpeI and PstI, while XylE gene was digested with XbaI and PstI.
the promoter and the XylE gene were gel purified.
a reaction between the promoter(still on vector) and XylE was set on for overnight ligation
PCR purification of GFP2 -> GFP construct ready for full fusion protein.
Gel purification of XylE lost DNA along the way. Thus PCR XylE1 with gradient for temperature scanning (taq): PCR round 1 included 62C and only rev primer to create pool of successful extensions with with rev primer (60-62). PCR round 2 with Fwd primer and temperature scale (68-68, 72-74).

Wednesday, 25th-Aug

Performed gel analysis on the purified XylE and J23101 to obtain ratios for ligation. First gel was scrapped as it produced appauling(explanation for Nick:really bad) results, 2nd gel run was successful.

Performed a ligation reaction between the vector containing J23101, and XylE(one on bench and one overnight one).
Transformation of the new plasmid into competent E.coli. Successfully transformed colonies can be selected for by loss of RFP expression.
XylE-1 PCR with temperature cascade. Gel analysis and purification.

Thursday, 26th-Aug

white colonies from the promoter-XylE transformed E.coli were picked and transferred to new amp plates. One is the replica plate and the other is the catechol assay plate.
XylE-1, two rounds of PCR/purification were run to obtain a sufficiently clear band. An additional PCR run for XylE-1 was discarded afterwards.

Friday, 27th-Aug

Catechol assay performed on promoter-XylE transformed E.coli. SUCCESSFULL

XylE-2 PCR and gel-purification cycles (2x) to obtain clear band. XylE-2 is now ready for assembly of the GFP-XylE fusion protein.

Saturday, 28th-Aug

Preparing the annealing step between the GFP-2 and XylE-2 constructs, we discovered sequence dissimilarities in the TEV-cleavable regions which we planned to use for the annealing step. Nevertheless a PCR was run with appropriate conditions (allowing for a minimal amount of unspecific annealing).

Sunday, 29th-Aug

Gel analysis of the attempted annealing reaction of GFP-2 XylE-2 showed unsufficiently clear bands for gel-purification. A new reaction is being prepared: 10 rounds of annealing PCR, followed by addition of primers (5' primer for GFP-2 and 3' primer for XylE-2) in order to introduce an amplification step in the reaction. --- Following Kirstins advice, we are discarding this reaction and wait for the arrival of new primers for XylE-2 (5' + TEV) and GFP-2 (3' +TEV).

Week 9

Week 9	Monday	Tuesday	Wednesday	Thursday	Friday
MORNING	mini-prep of promoter-XylE transformed E.coli	midi-prep of promoter-XylE transformed E.coli restriction digestion of pSB1C3+terminator vector DNA restriction digestion of promoter-XylE DNA	Gel purification kit of cut promoter-XylE DNA Preparation of M9 and LB medium for assay Gel analysis of of insert and vector DNA	find optimun wavelength for catechol assays	find optimun cell density and catechol concentration for assay
lunch break
AFTERNOON	gel analysis of mini-prepped promoter-XylE	PCR purification of cut pSB1C3+terminator vector DNA Gel analysis of cut promoter-XylE DNA	dephosphorylation of the vector+terminator ligation reaction between promoter-XylE insert and pSB1C3+terminator vector

Monday, 30th-Aug

Mini prep of 4 x J23101-XylE taken from 4 different colonies (8 14 24 27).
The gel analysis showed successful vector uptake.
Set up an overnight culture for midi using colony 24 (gel analysis showed similar results colony 24 was picked randomly.)

Tuesday, 31st-Aug

Midi prep of colony 24 for XylE-J23101 the final concentration was ~100ng/ul which wasnt so great but Chris says the protocol produces very poor yields.
We are performing the next building step of our vector. PSB1C3 containing terminator B0014 was cut with EcoRI and XbaI. The insert was cut with EcoRI and SpeI and both were incubated for 1.5hrs. Wolf is now running a gel to purify out the insert via gel purification and perform a PCR purification on the vector.
Advisors have decided it's best not to use Jeremy's tagged XylE due to the 93% difference. Kirsten will be tagging the registry XylE and we shall purify and assay with that instead.
We shall see purification expert Kieran tomorrow and talk through the process. Chris will also talk us through our characterization of XylE experiments- we will use the robot after it's been programmed but until then we can use the plate-reader.

Thursday, 2nd-Sept

Spectrophotometry experiments with XylE transformed E.coli in LB medium (M9 culture was contaminated) reveiled the followings: On catechol assay of the trasformed cells, the positive yellow output can be quantitively measured by a broad peak at 380nm.
Trasformation of competent E.coli cells with promoter-XylE-terminator pSB1C3vector.

Friday, 3rd-Sept

experiment to determine concentrations of catechol and cell density for assays

Week 10

Week 10	Monday	Tuesday	Wednesday	Thursday	Friday
MORNING	PCR extension of Pveg promoter	Gel purification of extended Pveg promoter	vector with insert tranformed into E.coli and plate to select for successful transformation	prepare overnight cultures of pVeg-RBS transformed E.coli	midi-prep of Pveg-RBS vector
lunch break
AFTERNOON		restriction digestion and gel purification of thr extended Pveg promoter overnight ligation of the cut Pveg promoter in a vector

Monday, 6th-Sept

PCR extension of Pveg promoter: EcoRI---Pveg---RBS-SpeI

IGEM:Imperial/2010/XylE team: Difference between revisions