Team:Edinburgh/Notebook/Red light producer

From 2010.igem.org

(Difference between revisions)
(→Red Light Producer)
(→Red Light Producer)
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*Template DNA used PyeaR  and P. pyralis luciferase BBa_K216015
*Template DNA used PyeaR  and P. pyralis luciferase BBa_K216015
*Purification done for 356K and 356R; stored in iGEM box (-20C).
*Purification done for 356K and 356R; stored in iGEM box (-20C).
-
 
-
 
-
 
-
 
-
 
[[File:07-09straight.JPG]]
[[File:07-09straight.JPG]]
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'''13/07/10'''
'''13/07/10'''
Goal of the day:  
Goal of the day:  
-
* red luciferase
+
 
-
**
+
Transformations:
Transformations:
*356R (2): one from 16 °C and one from room temp(RT)
*356R (2): one from 16 °C and one from room temp(RT)
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*S284T (2): one with 25/5 ligase, one with 1/7 ligase
*S284T (2): one with 25/5 ligase, one with 1/7 ligase
 +
* I had a burger for the lunch. It was very nice.
 +
* Now we are transforming cells:
 +
** 356R (2)& 356K (2): one form 16C, one from RT
 +
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/
 +
Empty edinbrick vector for control.
 +
* LuxAB + edinbrick ligation.
 +
* Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5.
'''22/07/10'''
'''22/07/10'''
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NaNO3 = 30mM Sodium Nitrate
NaNO3 = 30mM Sodium Nitrate
-
 
'''23/07/10'''
'''23/07/10'''
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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]
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'''
'''23/9/2010'''
* S248T-  
* S248T-  
-
**9.2; 5 (circled)
+
**9.2; 5  
-
** 9.4; 1 circled
+
** 9.4; 1  
**5; 4
**5; 4
-
 
* 356
* 356
** 3.3
** 3.3
-
 
* WT
* WT
-
** 3.2; 4 circled
+
** 3.2; 4  
-
** 1.4, 5 circled
+
** 1.4, 5

Revision as of 10:39, 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

9/7/2010

  • Ligations set up overnight- might have put too much ligase in 356K... Incubated at 15C.
  • Oure 356R and 356K are in the freezer- row 10 (G &H)?

13/07/10 Goal of the day:

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
  • I had a burger for the lunch. It was very nice.
  • Now we are transforming cells: 
** 356R (2)& 356K (2): one form 16C, one from RT
** S248T (2): 1 with 25/5 ligase, other with 1.7.10 ligase/

Empty edinbrick vector for control.

  • LuxAB + edinbrick ligation.
  • Using 100 ul transformed cells + 800 ul LB for all. Incubation started half 5.

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 Fujii et al, 2007 and the paper from Branchini et al, 2010

23/9/2010

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