Team:Edinburgh/Notebook/Red light producer

From 2010.igem.org

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Latest revision as of 02:11, 28 October 2010

These notes correspond to the part of the project detailed here.

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

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...

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...

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.

12/8/2010

Got the sequences for red luciferases
see: if we can detect Red Light with the luminometer?
Red Luciferases 356R and S284T were nNOT MUTANTS- just WT
set up ;iquid cultures with 30 ul nitrate for:
- 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate
- S284T - 5 from masterplate; 2, 4, 6 from masterplate
- 356K: 4 from masterplate

13/8/2010 Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE

16/8/2010

Sequecning of:
- WR1- 356R -use S284T forward primer
- WR2 -S284T -use 356 R reverse primer
- WR3- LovTap 03
- WR4 -LovTap 09

RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT

19/8/2010

star6: 56,869
star4: 13,471
356K: 263,778
star 2: 3,717
WT: 1,617832

Gel of the ligations: marker (lane 1), 356K (lane 2,3), S284T (lane 3,4), 358Rs (lane 5,6).

25/8/2010

Next step:- redo ligations for 356R; redo PCR for S284T
Ligations
Did MABEL PCR PPyInc + S284T primers
used 0.5 ul template DNA
Gel: marker (Lane1), ligation of PCR product for 356R (Lane2), Ligation of 07 PCR for S284T (lane3), Pure PCR from S284T.

26/8/2010

check the Fuji et. al paper, see what sequence they used to start off with.
check IAV in other luciferase
Fix scar before RBS and in teh second stop codon.
compared to WT, luciferase sequence has:
- N 50 P
- N 99 G
- SKL- IAV

27/8/2010

Ligations from yesterday out of freezer.
Running on gel
Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel).

Lane 1	Lane 2	Lane 3	Lane 4
Ladder	New S284T PCR - new ligase and the buffer	356 R ligation	S284T PCR + old ligase

30/8/2010

Primers have arrived for fixing the luciferase- removing 6 bases
Primers have arrived for up and downstream sequences of TnaA region.
Send Chairman sequence for T7 promoter
transformed 356R and S284T ligations from Friday
Used PCR product as a control
Plated out the transformed cells. Used 900 rather than 100 if possible

31/8/2010

We have S284T transformants but
- 14 colonies on ligation plate
- 4 colonies on control where we used PCR product instead of ligation

For sequencing-
- if sequencing a S284T mutant, use 356R/K reverse primer
- if sequencing 356R mutant, use S284T forward primer.

2/9/2010

minipreped 14 colonies (1-14)
run the gel with samples digested with EcoRI
here: samples 1-7

3/9/2010

S284T 8-14 (from yesterday)

8/9/2010

wWT of luciferase (left)
mutation of S284T (right)

9/9/2010

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5
Ladder	WT	S284T 1	luxCDE 9:4	LuxCDE 9:1

20/9/2010

send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)
all in psB1C3 vector.
Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS).
Run gel of 365K in psb1C3 of the digest. --> control: RFP + psb1C3
then add promoter

23/9/2010

Added in incubator room liquid culture ?) LacP + RBS + Luciferase for S284T, 356K and WT.
miniprep- 6 bottles
GLOWING

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

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+ul{ background-color: white; margin: 0; padding: 1.5em}
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+#menubar ul{ background: none; margin: 0.3em 0 0 1.5em; padding: 0}
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@@ Line 18: / Line 22: @@
 <div id="nav">
-<ul class="dropdown dropdown-horizontal">
+<ul class="dropdown dropdown-horizontal" style="background: none;">
   <li><a href="https://2010.igem.org/Team:Edinburgh" class="dir">home</a>
@@ Line 39: / Line 43: @@
     <li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li>
-    <li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">future work</a></li>
+    <li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">the future</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</a></li>
    </ul>
@@ Line 46: / Line 50: @@
   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a>
    <ul>
-    <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li>
+    <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the project</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li>
@@ Line 55: / Line 59: @@
     <li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li>
-    <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">future work</a></li>
+    <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">the future</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li>
    </ul>
@@ Line 66: / Line 70: @@
     <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">the bacterial model</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li>
+   <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li>
-    <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">future work</a></li>
+    <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">the future</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li>
    </ul>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a>
    <ul>
-    <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Aspects">human aspects</a></li>
+    <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li>
+   <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">iGEM survey</a></li>
+   <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Conversations">conversations</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li>
-    <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">results</a></li>
+    <li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li>
-    <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Future">future work</a></li>
+    <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">further thoughts</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li>
    </ul>
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    <ul>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li>
+   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li>
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     <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li>
+   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li>
+   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li>
     <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li>
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 <br>
+<div id="windowbox" style="border: .2em solid #660000; padding: 5px; position:fixed; top:50%; right:30px; width:8%;">
+<span style="color:ivory;">These <b>notes</b> correspond to the part of the <b>project</b> detailed <a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">here</a>.</span>
+</div>
 </html>
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 == Red Light Producer ==
 <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
+[[Image:151.JPG]]
+{| 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]]
+'''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...
+[[Image:157.jpg]]
+{| border="1"
+| 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...
+[[Image:113.JPG]]
+* 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'''
+[[Image:159.jpg]]
+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.
+'''12/8/2010'''
+* Got the sequences for red luciferases
+*see: if we can detect Red Light with the luminometer?
+* Red Luciferases 356R and S284T were nNOT MUTANTS- just WT
+* set up ;iquid cultures with 30 ul nitrate for:
+** 356R - no 3,1 from RT plate, no 5 from 16C plate; no 1 from Master plate
+** S284T - 5 from masterplate; 2, 4, 6 from masterplate
+** 356K: 4 from masterplate
+'''13/8/2010'''
+Big day today... TESTING YESTERDAYS PLAN- LUMINESCENCE
+'''16/8/2010'''
+* Sequecning of:
+** WR1- 356R -use S284T forward primer
+** WR2 -S284T -use 356 R reverse primer
+** WR3- LovTap 03
+**WR4 -LovTap 09
+* RESULTS: ONLY 356K WAS A REAL MUTANT; S284T AND 356R TURNED OUT TO BE STILL A WT
+'''19/8/2010'''
+* star6: 56,869
+* star4: 13,471
+* 356K: 263,778
+* star 2: 3,717
+* WT: 1,617832
+*Gel of the ligations: marker (lane 1), 356K (lane 2,3), S284T (lane 3,4), 358Rs (lane 5,6).
+[[Image:163.jpg]]
+'''25/8/2010'''
+* Next step:- redo ligations for 356R; redo PCR for S284T
+* Ligations
+*Did MABEL PCR PPyInc + S284T primers
+*used 0.5 ul template DNA
+*Gel: marker (Lane1), ligation of PCR product for 356R (Lane2), Ligation of 07 PCR for S284T (lane3), Pure PCR from S284T.
+[[Image:164.jpg]]
+'''26/8/2010'''
+* check the Fuji et. al paper, see what sequence they used to start off with.
+* check IAV in other luciferase
+* Fix scar before RBS and in teh second stop codon.
+* compared to WT, luciferase sequence has:
+** N 50 P
+** N 99 G
+** SKL- IAV
+[[Image:165.jpg]]
+'''27/8/2010'''
+* Ligations from yesterday out of freezer.
+* Running on gel
+* Column 3 9with 356R) has less DNA in it- not enough from ligation and messed up loading of gel).
+[[Image:132.JPG]]
+{| border="1"
+| Lane 1
+| Lane 2
+| Lane 3
+| Lane 4
+|-
+| Ladder
+| New S284T PCR - new ligase and the buffer
+| 356 R ligation
+| S284T PCR + old ligase
+|}
+'''30/8/2010'''
+* Primers have arrived for fixing the luciferase- removing 6 bases
+* Primers have arrived for up and downstream sequences of TnaA region.
+* Send Chairman sequence for T7 promoter
+* transformed 356R and S284T ligations from Friday
+* Used PCR product as a control
+* Plated out the transformed cells. Used 900 rather than 100 if possible
+'''31/8/2010'''
+* We have S284T transformants but
+** 14 colonies on ligation plate
+** 4 colonies on control where we used PCR product instead of ligation
+* For sequencing-
+** if sequencing a S284T mutant, use 356R/K reverse primer
+** if sequencing 356R mutant, use S284T forward primer.
+'''2/9/2010'''
+* minipreped 14 colonies (1-14)
+* run the gel with samples digested with EcoRI
+* here: samples 1-7
+[[Image:135.JPG]]
+'''3/9/2010'''
+* S284T 8-14 (from yesterday)
+[[Image:136.JPG]]
+'''8/9/2010'''
+* wWT of luciferase (left)
+* mutation of S284T (right)
+[[Image:168.jpg]]
+'''9/9/2010'''
+[[Image:170.jpg]]
+{| border="1"
+| Lane 1
+| Lane 2
+| Lane 3
+| Lane 4
+| Lane 5
+|-
+| Ladder
+| WT
+| S284T 1
+| luxCDE 9:4
+| LuxCDE 9:1
+|}
+'''20/9/2010'''
+*send for sequencing: 356K, S284T, WT (samples from 16/9: WT- 900, 2; also 16/9 S284T- 900, 4.)
+* all in psB1C3 vector.
+* Transform 356K and S284T with RBS and promoter (lacZ, LacI, RBS).
+* Run gel of 365K in psb1C3 of the digest.  --> control: RFP + psb1C3
+* then add promoter
+'''23/9/2010'''
+* Added in incubator room liquid culture  '''?)''' LacP + RBS + Luciferase for S284T, 356K and WT.
+* miniprep- 6 bottles
+* GLOWING
+* S248T-
+**9.2; 5
+** 9.4; 1
+**5; 4
+* 356
+** 3.3
+* WT
+** 3.2; 4
+** 1.4, 5