"

From 2010.igem.org

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 [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]

29/7/2010

• Double digest with EcoRI HF&PstI in buffer 2 (neb).
• 2 bands: with and without vector?
• did not leave the digest for too long...

Gel photo

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6
Ladder	S284T (4)	S284T (6)	356R (6, 900)	356R (6)	356K (4)

2/8/2010

• Red luciferases EcoRI HF&PstI double digests- lanes 2-7 in the second miniprep set.
• Ladder didn't work too well ,though...

GEL PIC

• Transform last years luciferase
• Look at papers for expression in E. coli
• Replace B0034 by stronger RBS?
• Confirm that we order bright luc.
• Single digest of constructs
• Test glowing with positive control

9/8/2010 Digest:

• WT1
• 356 R: (2)
• 356 K: (4)
• S284T: (6)
• (?)
• 3 eith EcoRI; 3 with EcoRi + PstI

10/8/2010

• Sequencing of red mutant luciferases.
• Forward primers for S284T and reverse for 356R and K should do.
• Concentration of primers for sequencing: 5uM.
• sequencing tubE: 6 ul liquid: 3ul DNA + 2ul water, 1 ul primer mix.
• when using miniprep DNA- remember to spin down the dirt- 1min, max speed, then take the DNA from the top to avoid disturbing any dirt that is settled.

23/9/2010

• S248T-
  • 9.2; 5
  • 9.4; 1
  • 5; 4
• 356
  • 3.3
• WT
  • 3.2; 4
  • 1.4, 5