Imperial College/7 September 2008

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=7th September 2008=
=7th September 2008=
==Wetlab==
==Wetlab==
 +
===''B.subtilis''===
 +
=====Monday=====
 +
*Prepare Lb agar plates and LB media for the week ahead.
 +
*Check PCR from friday on a 1% agarose gel.
 +
*Innoculate 20ml of LB agar for overnight culture
 +
=====Tuesday=====
 +
*Carry out the final protocol for the O.D.<sub>600</sub> vs cell number. From previous trials it is clear that it is better to base the data collection on the O.D.<sub>600</sub> as oppose to regular time intervals. In addition it is clear a greater spread of dilutions are required for an increased data collection.
 +
*Innoculate 20ml of LB agar for overnight culture
 +
 +
=====Wednesday=====
 +
*Microscope from 11-2 to collect wild-type movement of ''B.subtilis''
 +
*Count the cells from the O.D.<sub>600</sub> vs cell number experiment.
 +
 +
=====Thursday=====
 +
*Innoculate 20ml of LB agar for overnight culture
 +
 +
=====Friday=====
 +
*Microscope from 11-2 to collect wild-type movement of ''B.subtilis''
===Cloning===
===Cloning===

Revision as of 08:18, 8 September 2008

August
MTWTFSS
        [http://2008.igem.org/Imperial_College/1_August_2008 1] [http://2008.igem.org/Imperial_College/2_August_2008 2] [http://2008.igem.org/wiki/index.php?title=Imperial_College/3_August_2008&action=edit 3]
[http://2008.igem.org/Imperial_College/4_August_2008 4] [http://2008.igem.org/Imperial_College/5_August_2008 5] [http://2008.igem.org/wiki/index.php?title=Imperial_College/6_August_2008&action=edit 6] [http://2008.igem.org/Imperial_College/7_August_2008 7] [http://2008.igem.org/Imperial_College/8_August_2008 8] [http://2008.igem.org/wiki/index.php?title=Imperial_College/9_August_2008&action=edit 9] [http://2008.igem.org/Imperial_College/10_August_2008 10]
[http://2008.igem.org/Imperial_College/11_August_2008 11] [http://2008.igem.org/Imperial_College/12_August_2008 12] [http://2008.igem.org/Imperial_College/13_August_2008 13] [http://2008.igem.org/Imperial_College/14_August_2008 14] [http://2008.igem.org/Imperial_College/15_August_2008 15] [http://2008.igem.org/Imperial_College/16_August_2008 16] [http://2008.igem.org/Imperial_College/17_August_2008 17]
[http://2008.igem.org/Imperial_College/18_August_2008 18] [http://2008.igem.org/Imperial_College/19_August_2008 19] [http://2008.igem.org/Imperial_College/20_August_2008 20] [http://2008.igem.org/Imperial_College/21_August_2008 21] [http://2008.igem.org/Imperial_College/22_August_2008 22] [http://2008.igem.org/wiki/index.php?title=Imperial_College/23_August_2008&action=edit 23] [http://2008.igem.org/Imperial_College/24_August_2008 24]
[http://2008.igem.org/Imperial_College/25_August_2008 25] [http://2008.igem.org/Imperial_College/26_August_2008 26] [http://2008.igem.org/Imperial_College/27_August_2008 27] [http://2008.igem.org/Imperial_College/28_August_2008 28] [http://2008.igem.org/Imperial_College/29_August_2008 29] [http://2008.igem.org/wiki/index.php?title=Imperial_College/30_August_2008&action=edit 30] [http://2008.igem.org/Imperial_College/31_August_2008 31]
September
MTWTFSS
[http://2008.igem.org/Imperial_College/1_September_2008 1] [http://2008.igem.org/Imperial_College/2_September_2008 2] [http://2008.igem.org/Imperial_College/3_September_2008 3] [http://2008.igem.org/Imperial_College/4_September_2008 4] [http://2008.igem.org/Imperial_College/5_September_2008 5] [http://2008.igem.org/wiki/index.php?title=Imperial_College/6_September_2008&action=edit 6] [http://2008.igem.org/Imperial_College/7_September_2008 7]
[http://2008.igem.org/Imperial_College/8_September_2008 8] [http://2008.igem.org/Imperial_College/9_September_2008 9] [http://2008.igem.org/Imperial_College/10_September_2008 10] [http://2008.igem.org/Imperial_College/11_September_2008 11] [http://2008.igem.org/Imperial_College/12_September_2008 12] [http://2008.igem.org/wiki/index.php?title=Imperial_College/13_September_2008&action=edit 13] [http://2008.igem.org/wiki/index.php?title=Imperial_College/14_September_2008&action=edit 14]
[http://2008.igem.org/Imperial_College/15_September_2008 15] [http://2008.igem.org/Imperial_College/16_September_2008 16] [http://2008.igem.org/Imperial_College/17_September_2008 17] [http://2008.igem.org/Imperial_College/18_September_2008 18] [http://2008.igem.org/Imperial_College/19_September_2008 19] [http://2008.igem.org/wiki/index.php?title=Imperial_College/20_September_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=Imperial_College/21_September_2008&action=edit 21]
[http://2008.igem.org/Imperial_College/22_September_2008 22] [http://2008.igem.org/Imperial_College/23_September_2008 23] [http://2008.igem.org/Imperial_College/24_September_2008 24] [http://2008.igem.org/Imperial_College/25_September_2008 25] [http://2008.igem.org/Imperial_College/26_September_2008 26] [http://2008.igem.org/wiki/index.php?title=Imperial_College/27_September_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=Imperial_College/28_September_2008&action=edit 28]
[http://2008.igem.org/Imperial_College/29_September_2008 29] [http://2008.igem.org/Imperial_College/30_September_2008 30]
October
MTWTFSS
    [http://2008.igem.org/Imperial_College/1_October_2008 1] [http://2008.igem.org/Imperial_College/2_October_2008 2] [http://2008.igem.org/wiki/index.php?title=Imperial_College/3_October_2008&action=edit 3] [http://2008.igem.org/wiki/index.php?title=Imperial_College/4_October_2008&action=edit 4] [http://2008.igem.org/wiki/index.php?title=Imperial_College/5_October_2008&action=edit 5]
[http://2008.igem.org/wiki/index.php?title=Imperial_College/6_October_2008&action=edit 6] [http://2008.igem.org/Imperial_College/7_October_2008 7] [http://2008.igem.org/wiki/index.php?title=Imperial_College/8_October_2008&action=edit 8] [http://2008.igem.org/Imperial_College/9_October_2008 9] [http://2008.igem.org/wiki/index.php?title=Imperial_College/10_October_2008&action=edit 10] [http://2008.igem.org/wiki/index.php?title=Imperial_College/11_October_2008&action=edit 11] [http://2008.igem.org/wiki/index.php?title=Imperial_College/12_October_2008&action=edit 12]
[http://2008.igem.org/Imperial_College/13_October_2008 13] [http://2008.igem.org/wiki/index.php?title=Imperial_College/14_October_2008&action=edit 14] [http://2008.igem.org/Imperial_College/15_October_2008 15] [http://2008.igem.org/Imperial_College/16_October_2008 16] [http://2008.igem.org/Imperial_College/17_October_2008 17] [http://2008.igem.org/wiki/index.php?title=Imperial_College/18_October_2008&action=edit 18] [http://2008.igem.org/wiki/index.php?title=Imperial_College/19_October_2008&action=edit 19]
[http://2008.igem.org/wiki/index.php?title=Imperial_College/20_October_2008&action=edit 20] [http://2008.igem.org/wiki/index.php?title=Imperial_College/21_October_2008&action=edit 21] [http://2008.igem.org/wiki/index.php?title=Imperial_College/22_October_2008&action=edit 22] [http://2008.igem.org/wiki/index.php?title=Imperial_College/23_October_2008&action=edit 23] [http://2008.igem.org/wiki/index.php?title=Imperial_College/24_October_2008&action=edit 24] [http://2008.igem.org/wiki/index.php?title=Imperial_College/25_October_2008&action=edit 25] [http://2008.igem.org/wiki/index.php?title=Imperial_College/26_October_2008&action=edit 26]
[http://2008.igem.org/wiki/index.php?title=Imperial_College/27_October_2008&action=edit 27] [http://2008.igem.org/wiki/index.php?title=Imperial_College/28_October_2008&action=edit 28] [http://2008.igem.org/wiki/index.php?title=Imperial_College/29_October_2008&action=edit 29] [http://2008.igem.org/wiki/index.php?title=Imperial_College/30_October_2008&action=edit 30] [http://2008.igem.org/wiki/index.php?title=Imperial_College/31_October_2008&action=edit 31]

7th September 2008

Wetlab

B.subtilis

Monday
  • Prepare Lb agar plates and LB media for the week ahead.
  • Check PCR from friday on a 1% agarose gel.
  • Innoculate 20ml of LB agar for overnight culture
Tuesday
  • Carry out the final protocol for the O.D.600 vs cell number. From previous trials it is clear that it is better to base the data collection on the O.D.600 as oppose to regular time intervals. In addition it is clear a greater spread of dilutions are required for an increased data collection.
  • Innoculate 20ml of LB agar for overnight culture
Wednesday
  • Microscope from 11-2 to collect wild-type movement of B.subtilis
  • Count the cells from the O.D.600 vs cell number experiment.
Thursday
  • Innoculate 20ml of LB agar for overnight culture
Friday
  • Microscope from 11-2 to collect wild-type movement of B.subtilis

Cloning

Ongoing/Where there is spare time
  • PCR trial all genomic sequences to determine optimal conditions with Taq then produce clones with Pfu
  • Digest with XbaI and SpeI and incorporate into biobricks
  • Follow up by assaying orientation (either with sequence internal restriction site or by using XbaI and SpeI, as they should only be able to cut inserts in the correct orientation)
Monday
  • GeneArt constructs 7, 8, 9 and 11 (SacB-EAK16, LipA-EAK16, P43-RBSgsiB and Pveg-RBSgsiB) to be ligated into Biobrick vector pSB1A2 or pSB1AK3
  • CAT to be ligated togethre with the terminator (B0015) to form CAT-Terminator
  • AmyE 5' and 3' integration sequences and Aad9 (Spectinomycin resistance gene) to be integrated into pSB1A2 or pSB1AK3
  • GeneArt constructs 3, 6, 10 and 12 (EpsE gene and PgsiB-RBSspoVG in construct 3, LipA-Elastin and PgsiB-RBSgsiB in construct 6, P43-RBSspoVG and Pveg-RBSspoVG), along with all above biobricks will be transformed into Xl1-Blue E.coli
  • Depending on results, LacI PCR to be digested and purified ready for ligation into pSB1A2/pSB1AK3


Tuesday
  • E.coli containing GeneArt Constructs 7, 8, 9, 11 and CAT-Terminator to be grown up for midi-prepping Wednesdays
  • E.coli containing AmyE 5' and 3' integration sequences and Aad9 to be grown for mini-prep on Wednesday
  • GFP-Terminator to be cut ready for building
  • Ligate LacI into biobrick and transform into XL1-Blue
Wednesday
  • Constructs 7, 8, 9, 11 and CAT-Terminator to be midi-prepped and digested ready for building of the phase 1 constructs
  • E.coli containing constructs 10 and 12 to be grown up overnight for midi-prepping
  • Aad9 and the AmyE integration sequences to be miniprepped and digested to check insert orientation
  • Correctly orientated biobricks to be grown up overnight for midi-prepping Thursday
  • Grow E.coli containing the new LacI biobrick
  • Time permitting (If not will be carried out Thursday):
    • CAT-Terminator to be ligated to P43-RBSgsiB (9) and Pveg-RBSgsiB (11) separately OR AmyE 3' integration sequence tomorrow (depends on status of AmyE 3' sequence) and transformed into XL1-BlueE.coli
    • GFP-Terminator to be ligated to P43-RBSgsiB (9) and Pveg-RBSgsiB (11) separately and transformed into XL1-BlueE.coli(depending on CAT-Terminator ligation petner)
Thursday
  • Constructs 10, 12 the AmyE integration sequences and Aad9 to be midiprepped
  • AmyE integration sequences and Aad9 to be digested ready for builiding of phase 1 construct
  • Constructs 10 and 12 to be digested and gel-purified ready to be incorporated into biobricks
  • E.coli containing constructs 3 and 6 to be grown up overnight for midi-prepping
  • Mini-prep the LacI biobrick and check for correct orientation. Grow a culture with the LacI gene in the correct orientation for midi-prep tomorrow
  • Time permitting (If not may be carried out Friday):
    • Aad9 to be ligated to the P43-RBSgsiB and Pveg-RBSgsiB separately and transformed into XL1-BlueE.coli
    • Terminator to be ligated AmyE integration sequeces (separately) and transformed into XL1-BlueE.coli
    • Constructs 10 and 12 to be ligated into biobricks
Friday
  • Constructs 3 and 6 to be midiprepped
  • Constructs 3 and 6 digested and run on a gel to separate components by gel purification
  • Midi-prep LacI
  • Finish any unfinished jobs from earlier in week

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