IGEM:Imperial/2010/XylE team

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(added 26th,27th, 28th, 29th for fusion protein construction)
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*Performed a ligation reaction between the vector containing J23101, and XylE(one on bench and one overnight one).  
*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.
*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==
==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.
*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.
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* 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==
==Friday, 27th-Aug==
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*Catechol assay performed on promoter-XylE transformed E.coli. <font color = green>'''SUCCESSFULL'''</font>
*Catechol assay performed on promoter-XylE transformed E.coli. <font color = green>'''SUCCESSFULL'''</font>
[[Image:Catechol Assay before.jpg|thumb|left|Plate before adding catechol assay]] [[Image:Catechol assay after (27-8).jpg|thumb|center|After addission of catechol colonies turn yellow-orange in seconds!!]]
[[Image:Catechol Assay before.jpg|thumb|left|Plate before adding catechol assay]] [[Image:Catechol assay after (27-8).jpg|thumb|center|After addission of catechol colonies turn yellow-orange in seconds!!]]
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* 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.
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 +
==Saturday, 28th-Aug==
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 +
* 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).
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 +
==Sunday, 29th-Aug==
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* 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).

Revision as of 05:59, 31 August 2010

Image:Logo.JPG Image:Txeee.JPG‎

Contents

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 6 Monday Tuesday Wednesday Thursday Friday Weekend
MORNING
  • mini-prep kit of XylE-transformed E.coli (already overnight grown)
lunch break
AFTERNOON
  • Starting of “Testing expression of XylE in E.coli” objective
  • 1)Annealing EcoRI and speI oligos to J23101 promoter which will be annealed later in front of the RBS-XylE registry gene (overnight)
  • gel analysis of mini-prep derived XylE plasmid.(requires first digestion of the vector with restriction enzymes)

Week 7

Week 7 Monday Tuesday Wednesday Thursday Friday Weekend
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

Week 8

Week 6 Monday Tuesday Wednesday Thursday Friday Weekend
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

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.

Image:10-08-13 Digest image.jpg

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
Midi-prep XylE digestion with xbaI and PstI The smaller size bands at lanes 2 & 3 are the ones that are going to be cut out and used in gel purification to extract the XylE gene (with sticky ends for XbaL and PstI).
Midi-prep XylE digestion with xbaI and PstI The smaller size bands at lanes 2 & 3 are the ones that are going to be cut out and used in gel purification to extract the XylE gene (with sticky ends for XbaL and PstI).
  • 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
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
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

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
Plate before adding catechol assay
Plate before adding catechol assay
After addission of catechol colonies turn yellow-orange in seconds!!
After addission of catechol colonies turn yellow-orange in seconds!!
  • 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).

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