Team:Edinburgh/Notebook/Red light producer

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(→Red Light Producer)
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== Red Light Producer ==
== Red Light Producer ==
<br>
<br>
 +
 +
'''29/6/10'''
 +
 +
Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)
 +
 +
'''8/7/2010'''
 +
* Red luciferase mutagenesis primers arrived- 3 sets:
 +
** S248T
 +
** 356 K
 +
** 356 R
 +
 +
* Followed the protocol with 34ul water + 0.5 template DNA
 +
* Put the primers and template in green box '''(???)'''
 +
*Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer
 +
 +
'''GEL PHOTO'''
 +
 +
{| border="1"
 +
| Marker
 +
| S284T
 +
| 356K
 +
| 356R
 +
|}
 +
 +
 +
[[File:07-08.JPG]]
 +
 +
 +
*356K and 356R look fine
 +
*going to run S284T at a higher ** temp tomorrow to see if it works better
 +
*all PCR tubes stored in freezer
 +
 +
 +
 +
'''09/7/10'''
 +
 +
Redo PCR of Red Luciferase
 +
*Everything except template DNA + Kod polymerase kept on ice
 +
*Ran PCR
 +
*Template DNA used PyeaR  and P. pyralis luciferase BBa_K216015
 +
*Purification done for 356K and 356R; stored in iGEM box (-20C).
 +
 +
 +
 +
 +
 +
 +
[[File:07-09straight.JPG]]
 +
 +
 +
'''13/07/10'''
 +
 +
Transformations:
 +
*356R (2): one from 16 °C and one from room temp(RT)
 +
*356K (2): one from 16 °C and one from room temp(RT)
 +
*S284T (2): one with 25/5 ligase, one with 1/7 ligase
 +
 +
 +
'''22/07/10'''
 +
 +
Liquid Cultures (amp 80):
 +
*356R (RT) – 1 with NaNO3 and 1 without
 +
**(16°C) – 2 with NaNO3 and 2 without
 +
*356K (RT) – 1 with NaNO3 and 1 without
 +
**(16°C) – 2 with NaNO3 and 2 without
 +
*S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without
 +
**(900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without
 +
**(900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without
 +
 +
NaNO3 = 30mM Sodium Nitrate
 +
 +
 +
'''23/07/10'''
 +
 +
Double digest of K098010
 +
 +
28/07/10
 +
 +
Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf  Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]
 +
 +
 +
'''23/9/2010'''
 +
 +
* S248T-
 +
**9.2; 5 (circled)
 +
** 9.4; 1 circled
 +
**5; 4
 +
 +
* 356
 +
** 3.3
 +
 +
* WT
 +
** 3.2; 4 circled
 +
** 1.4, 5 circled

Revision as of 10:16, 6 October 2010





Red Light Producer


29/6/10

Now preparing primers to make a set of different spectra bioluminescent proteins. Need to order primers for Site directed mutagenesis of the Firely luciferase to make it red (MABEL protocol), and to make click beetle luciferase Rluc8 (which emits light at about 420nm in presence of bisdeoxycoelenterazine - about the same price a coelenterazine though)

8/7/2010

  • Red luciferase mutagenesis primers arrived- 3 sets:
    • S248T
    • 356 K
    • 356 R
  • Followed the protocol with 34ul water + 0.5 template DNA
  • Put the primers and template in green box (???)
  • Ran Gel: 5 µL PCR product, 5 µL water, 2.5 µL buffer

GEL PHOTO

Marker S284T 356K 356R


File:07-08.JPG


  • 356K and 356R look fine
  • going to run S284T at a higher ** temp tomorrow to see if it works better
  • all PCR tubes stored in freezer


09/7/10

Redo PCR of Red Luciferase

  • Everything except template DNA + Kod polymerase kept on ice
  • Ran PCR
  • Template DNA used PyeaR and P. pyralis luciferase BBa_K216015
  • Purification done for 356K and 356R; stored in iGEM box (-20C).




File:07-09straight.JPG


13/07/10

Transformations:

  • 356R (2): one from 16 °C and one from room temp(RT)
  • 356K (2): one from 16 °C and one from room temp(RT)
  • S284T (2): one with 25/5 ligase, one with 1/7 ligase


22/07/10

Liquid Cultures (amp 80):

  • 356R (RT) – 1 with NaNO3 and 1 without
    • (16°C) – 2 with NaNO3 and 2 without
  • 356K (RT) – 1 with NaNO3 and 1 without
    • (16°C) – 2 with NaNO3 and 2 without
  • S284T (100 µL and 25/5 ligase) – 2 with NaNO3 and 2 without
    • (900 µL and 25/5 ligase) – 3 with NaNO3 and 3 without
    • (900 µL and 1/7 ligase) – 5 with NaNO3 and 5 without

NaNO3 = 30mM Sodium Nitrate


23/07/10

Double digest of K098010

28/07/10

Primer design to produce a stable red light luciferase. The mutations for increasing bioluminescence come from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4NHM57C-1&_user=809099&_coverDate=07%2F15%2F2007&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414502247&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=00d9c92f6452bd276c6ea606ed7d3bbf Fujii et al, 2007] and the paper from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4X6MSYB-7&_user=809099&_coverDate=01%2F15%2F2010&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1414503636&_rerunOrigin=google&_acct=C000043939&_version=1&_urlVersion=0&_userid=809099&md5=297be52832193b4d026953632a1edf3c Branchini et al, 2010]


23/9/2010

  • S248T-
    • 9.2; 5 (circled)
    • 9.4; 1 circled
    • 5; 4
  • 356
    • 3.3
  • WT
    • 3.2; 4 circled
    • 1.4, 5 circled
Retrieved from "http://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer"