Team:Queens/Notebook

From 2009.igem.org

(Difference between revisions)
Line 80: Line 80:
-RFP
-RFP
-E1010 <i>Plate 1, Well 18F, plasmid pSB2K3 (K)</i>
-E1010 <i>Plate 1, Well 18F, plasmid pSB2K3 (K)</i>
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">17/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Transformed the following parts into Top10
 +
1. K157013 <i>Plate 3, Well 3G, K157000, Res:A</i>
 +
2. I712078 <i>Plate 2, well 14M, J70003, Res:A</i>
 +
3. I712077 <i>Plate 2, Well 14K, J70003, Res:A</i>
 +
4. R0062 <i>Plate 1, Well 6O, pSB1A2, Res:A</i>
 +
5. R1062 <i>Plate 1, Well 8G, pSB1A2, Res:A</i>
 +
6. K098910 <i>Plate 3, Well 11N, pSB3K3, Res:A</i>
 +
7. B0015 <i>Plate 1, Well 23L, pSB1AK3, Res:AK</i>
 +
8. E1010 <i>Plate 2, Well 18F, pSB2K3, Res:K</i>
 +
-Left in 37C for four hours and left on lab bench at room temperature overnight
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">18/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-No colonies were observered on the plates
 +
-Left the plates in 37C for four hours
 +
-Picked one colony from each plate and made overnight broth culture for glycerol stock.
 +
-K098010 plate did not grow, so it was left in 37C overnight.
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">19/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Made the glycerol stocks from the overnight broth cultures
 +
-Started broth culture for K098010
 +
-Re-transformed the E1010 in Top10
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">22/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Restarted the broth culture for K098010 because it was overgrown (already in stationary phase)
 +
-Observed no colonies on E1010 plate. Discovered that E1010 should be on Kanamycin resistance plate.
 +
 +
Things to Do:
 +
-Get out the standard plasmid backbones
 +
-High copy number assembly plasmid backbone
 +
-pSB1A3 <i>Plate 1, Well 1K</i>
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">23/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Submitted VLA construct to Mr. GENE for synthesis
 +
-Placed orders for PCR primers
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">24/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Transformed the following parts into Top10
 +
9.  RBS-LuxR J37033 <i>Plate 3, Well 4O, pSB1A2, Res:A</i>
 +
10. RBS-LuxL-ter F1610 <i>Plate 2, Well 24G, pSB1AK3, Res:AK</i>
 +
11. RBS-LuxL K081008 <i>Plate 2, Well 10L, pSB1A2, Res:A</i>
 +
12. RBS-LuxR-ter I0462 <i>Plate 1, Well 8O, pSB1A2, Res:A</i>
 +
13. Pconst J23119 <i>Plate 1, Well 18A, pSB1A2, Res:A</i>
 +
14. LuxR C0062 <i>Plate 1, Well 4O, pSB1A2, Res:A</i>
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">25/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Purchased QiaMiniPrep Kit from BioBar
 +
-Picked colonies from plates and re-cultured in broth; left overnight
 +
-Ordered ITGA4 cDNA plasmid from OpenBioSystem
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">26/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Made glycerol stocks for parts transformed 23/06/2009
 +
-Designed primer for PCR out VLA cDNA plasmid and primer for pLux-RBS-HindIII
 +
-Transformed the following parts into Top10 and left the plates in 37C overnight.
 +
15. RBS B0034 <i>Plate 1, Well 2M, pSB1A2, Res:A</i>
 +
16. HemeC I716154 <i>Plate 1, Well 17B, pSB1A2, Res:A</i>
 +
17. HemeD I716155 <i>Plate 1, Well 17D, pSB1A2, Res:A</i>
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">27/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Picked colonies from plates and made glycerol stocks
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">28/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Transformed gycerol stocks into broth culture
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">29/06/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Purified plasmids containing parts 1-17 from Top10 using Qiagen Spin MiniPrep Kit.
 +
-Performed Agarose Gel Electrophoresis to check the size of the parts. Bends did
 +
not migrate very far, possible due tot he fact that circular plasmids migrate very
 +
slowly. We decided to digest the plasmids with a BioBrick restriction enzyme
 +
and then run the gel again.
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">02/07/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Re-ran the Agarose Gel Electrophoresis to ckeck the size of the parts. It worked.
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">06/07/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Transformed the following parts into Top10
 +
18. GFP E0040 <i>Plate 1, Well 14K, pSB1A2, Res:A</i>
 +
19. GFP constr. E0840 <i>Plate 1, Well 12O, pSB1A2, Res:A</i>
 +
20. pTet+GFP I13522 <i>Plate 2, Well 8A, pSB1A2, Res:A</i>
 +
21. LuxR constr. K091204 <i>Plate 2, Well 8J, pSB1A2, Res:A</i>
 +
22. LuxL+GFP J37034 <i>Plate 2, Well 7I, pSB1A2, Res:AK</i>
 +
23. pSB1AC3 <i>Plate 1, Well 11A Res:AC</i>
 +
24. pSB1AK3 <i>Plate 1, Well 13A Res:AK</i>
 +
25. pSB1AT3 <i>Plate 1, Well 15A, Res:AT</i>
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">07/07/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Picked colonies from plates and made broth culture which was left to grow overnight
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">08/07/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Purified the plasmids of parts 18-25 using Qiagen Spin MiniPrep Kit.
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">09/07/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Ran a 1% Agarose gel electrophoresis to confirm the plasmid lengths of parts 18 to 25
 +
-Gel loading and concentrations:
 +
I. E0040 120μg/μL loaded 12μL
 +
II. pSB1AC3 120μg/μL loaded 12μL
 +
III. R0062 120μg/μL loaded 12μL
 +
IV. pSB1AT3 120μg/μL loaded 12μL
 +
V. J23119 120μg/μL loaded 12μL
 +
VI. J37034 120μg/μL loaded 12μL
 +
VII. K091204 120μg/μL loaded 12μL
 +
VIII. E0840 120μg/μL loaded 10μL
 +
IX. B0034 50μg/μL loaded 10μL
 +
X. F1610 50μg/μL loaded 10μL
 +
XI. I13522 120μg/μL loaded 10μL
 +
-Calculated the relative concentrations of the parts by comparing the bands with
 +
the ladder (The 5000bp ladder is about 120ng/μL)
 +
-Parts digested:
 +
I. R0062 Plux prefix EcoR1+Spe1
 +
II. pSB1AT3 backbone EcoR1+Pst1
 +
III. J23119 Pconst prefix EcoR1+Spe1
 +
IV. B0034 RBS suffix Xba1+Pst1
 +
V. F1610 RBS-LuxI-STOP suffix Xba1+Pst1
 +
VI. B0015 Terminator prefix EcoR1+Spe1
 +
-Restriction digestion mix recipe:
 +
-600ng of DNA
 +
-4μL restriction buffer
 +
-0.5μL EcoR1 and 0.5μL Spe1, or
 +
-0.5μL Xba1 and 0.5μL Pst1
 +
-Up to 35μL of ddH<sub>2</sub>0
 +
-Digested the parts using corresponding BioBrick restriction enzymes, and then
 +
purified parts using QiaQuick PCR Purification Kit.
 +
-Stored the purified DNA in -20C overnight
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">10/07/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Ran a 1% Agarose gel electrophoresis to confirm the products of restriction
 +
digestions.
 +
-The expected bands of the parts did not show up. This is probably dude to
 +
the fact that most of the parts have lengths within 100bp and the QiaQuick
 +
PCR purification kit removes DNA below 100bp length.
 +
-The miniprep plasmids of the parts are digested again with corresponding
 +
restriction enzymes for 2 hours
 +
-Enzymes are deactivated (denatured) by heating at 65C
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">13/07/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Ligated the parts into the following constructs using the <i>T4 Ligase Protocol</i>
 +
-P<sub>const</sub> - RBS - LuxL - 2xSTOP
 +
-P<sub>lux</sub> - RBS
 +
-P<sub>const</sub> - RBS (Heme Oxygenase)
 +
-Terminator - RBS
 +
-Made broth culture from the VLA cDNA glycerol stock
 +
</pre>
 +
</p>
 +
 +
 +
<p style="font-size:120%;font-family:palatino linotype;color:#172C4E">14/07/2009</p>
 +
<p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
<pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
 +
-Purified the plasmid for ITGA4 using QiaMiniPrep Spin Kit
 +
-Recultured ITGA4-containing cells in broth
</pre>
</pre>
</p>
</p>

Revision as of 13:58, 16 July 2009



Menu

Laboratory One: Harry, Bogdan, James, Bryant

Laboratory Two: Kate, Mike

Tech Support and Laboratory Three: Parthiv, Chris, Chris



Laboratory One: Harry, Bogdan, James, Bryant


16/06/2009

-Lab Meeting
-Finished the sequence for the surface expression construct
-Finalized to-do list
-Mini-prepped the following:
	-Linker K157013 Plate 3, Well 3G, plasmid Bba_K157000(A)
	-TEV Protease
		-I712078 (C-Terminus) Plate 2, Well 14M, plasmid J70003 (A)
		-I712077 (N-Terminus) Plate 2, Well 14K, plasmid J70003 (A)
	-pLux
		-R0062 (not leaky) Plate 1, Well 6O, plasmid pSB1A2 (A)
		-R1062 (median strength in the absence of luxR/HSL)
		 Plate 1, Well 8G, plasmid pSB1A2 (A)
	-HO-pcyA
		-K098010 Plate 3, Well 11N, plasmid pSB3k3 (K)
	-Terminator
		-B0015 Plate 1, Well 23L, plasmid pSB1AK3 (AK)
	-RFP
		-E1010 Plate 1, Well 18F, plasmid pSB2K3 (K)

17/06/2009

-Transformed the following parts into Top10
	1. K157013	Plate 3, Well 3G, K157000, Res:A
	2. I712078	Plate 2, well 14M, J70003, Res:A
	3. I712077	Plate 2, Well 14K, J70003, Res:A
	4. R0062	Plate 1, Well 6O, pSB1A2, Res:A
	5. R1062	Plate 1, Well 8G, pSB1A2, Res:A
	6. K098910	Plate 3, Well 11N, pSB3K3, Res:A
	7. B0015	Plate 1, Well 23L, pSB1AK3, Res:AK
	8. E1010	Plate 2, Well 18F, pSB2K3, Res:K
-Left in 37C for four hours and left on lab bench at room temperature overnight

18/06/2009

-No colonies were observered on the plates
-Left the plates in 37C for four hours
-Picked one colony from each plate and made overnight broth culture for glycerol stock.
-K098010 plate did not grow, so it was left in 37C overnight.

19/06/2009

-Made the glycerol stocks from the overnight broth cultures
-Started broth culture for K098010
-Re-transformed the E1010 in Top10

22/06/2009

-Restarted the broth culture for K098010 because it was overgrown (already in stationary phase)
-Observed no colonies on E1010 plate. Discovered that E1010 should be on Kanamycin resistance plate.

Things to Do:
-Get out the standard plasmid backbones
-High copy number assembly plasmid backbone
	-pSB1A3 Plate 1, Well 1K

23/06/2009

-Submitted VLA construct to Mr. GENE for synthesis
-Placed orders for PCR primers

24/06/2009

-Transformed the following parts into Top10
	9.  RBS-LuxR		J37033	Plate 3, Well 4O, pSB1A2, Res:A
	10. RBS-LuxL-ter	F1610	Plate 2, Well 24G, pSB1AK3, Res:AK
	11. RBS-LuxL		K081008	Plate 2, Well 10L, pSB1A2, Res:A
	12. RBS-LuxR-ter	I0462	Plate 1, Well 8O, pSB1A2, Res:A
	13. Pconst		J23119	Plate 1, Well 18A, pSB1A2, Res:A
	14. LuxR		C0062	Plate 1, Well 4O, pSB1A2, Res:A

25/06/2009

-Purchased QiaMiniPrep Kit from BioBar
-Picked colonies from plates and re-cultured in broth; left overnight
-Ordered ITGA4 cDNA plasmid from OpenBioSystem

26/06/2009

-Made glycerol stocks for parts transformed 23/06/2009
-Designed primer for PCR out VLA cDNA plasmid and primer for pLux-RBS-HindIII
-Transformed the following parts into Top10 and left the plates in 37C overnight.
	15. RBS		B0034	Plate 1, Well 2M, pSB1A2, Res:A
	16. HemeC	I716154	Plate 1, Well 17B, pSB1A2, Res:A
	17. HemeD	I716155	Plate 1, Well 17D, pSB1A2, Res:A

27/06/2009

-Picked colonies from plates and made glycerol stocks

28/06/2009

-Transformed gycerol stocks into broth culture

29/06/2009

-Purified plasmids containing parts 1-17 from Top10 using Qiagen Spin MiniPrep Kit.
-Performed Agarose Gel Electrophoresis to check the size of the parts. Bends did
 not migrate very far, possible due tot he fact that circular plasmids migrate very
 slowly. We decided to digest the plasmids with a BioBrick restriction enzyme
 and then run the gel again.

02/07/2009

-Re-ran the Agarose Gel Electrophoresis to ckeck the size of the parts. It worked.

06/07/2009

-Transformed the following parts into Top10
	18. GFP			E0040	Plate 1, Well 14K, pSB1A2, Res:A
	19. GFP constr.		E0840	Plate 1, Well 12O, pSB1A2, Res:A
	20. pTet+GFP		I13522	Plate 2, Well 8A, pSB1A2, Res:A
	21. LuxR constr.	K091204	Plate 2, Well 8J, pSB1A2, Res:A
	22. LuxL+GFP		J37034	Plate 2, Well 7I, pSB1A2, Res:AK
	23. pSB1AC3			Plate 1, Well 11A Res:AC
	24. pSB1AK3			Plate 1, Well 13A Res:AK
	25. pSB1AT3			Plate 1, Well 15A, Res:AT

07/07/2009

-Picked colonies from plates and made broth culture which was left to grow overnight

08/07/2009

-Purified the plasmids of parts 18-25 using Qiagen Spin MiniPrep Kit.

09/07/2009

-Ran a 1% Agarose gel electrophoresis to confirm the plasmid lengths of parts 18 to 25
-Gel loading and concentrations:
	I.	E0040		120μg/μL	loaded 12μL 
	II.	pSB1AC3		120μg/μL 	loaded 12μL
	III.	R0062		120μg/μL	loaded 12μL
	IV.	pSB1AT3		120μg/μL	loaded 12μL
	V.	J23119		120μg/μL	loaded 12μL
	VI.	J37034		120μg/μL	loaded 12μL
	VII.	K091204		120μg/μL	loaded 12μL
	VIII.	E0840		120μg/μL	loaded 10μL
	IX.	B0034		50μg/μL		loaded 10μL
	X.	F1610		50μg/μL		loaded 10μL
	XI.	I13522		120μg/μL	loaded 10μL
-Calculated the relative concentrations of the parts by comparing the bands with
 the ladder (The 5000bp ladder is about 120ng/μL)
-Parts digested:
	I.	R0062 		Plux 		prefix		EcoR1+Spe1
	II.	pSB1AT3				backbone	EcoR1+Pst1
	III.	J23119		Pconst		prefix		EcoR1+Spe1
	IV.	B0034		RBS		suffix 		Xba1+Pst1
	V.	F1610		RBS-LuxI-STOP	suffix		Xba1+Pst1
	VI.	B0015		Terminator 	prefix 		EcoR1+Spe1
-Restriction digestion mix recipe:
	-600ng of DNA
	-4μL restriction buffer
	-0.5μL EcoR1 and 0.5μL Spe1, or
	-0.5μL Xba1 and 0.5μL Pst1
	-Up to 35μL of ddH20
-Digested the parts using corresponding BioBrick restriction enzymes, and then
 purified parts using QiaQuick PCR Purification Kit.
-Stored the purified DNA in -20C overnight

10/07/2009

-Ran a 1% Agarose gel electrophoresis to confirm the products of restriction 
 digestions.
-The expected bands of the parts did not show up. This is probably dude to
 the fact that most of the parts have lengths within 100bp and the QiaQuick
 PCR purification kit removes DNA below 100bp length.
-The miniprep plasmids of the parts are digested again with corresponding
 restriction enzymes for 2 hours
-Enzymes are deactivated (denatured) by heating at 65C

13/07/2009

-Ligated the parts into the following constructs using the T4 Ligase Protocol
	-Pconst - RBS - LuxL - 2xSTOP
	-Plux - RBS
	-Pconst - RBS (Heme Oxygenase)
	-Terminator - RBS
-Made broth culture from the VLA cDNA glycerol stock

14/07/2009

-Purified the plasmid for ITGA4 using QiaMiniPrep Spin Kit
-Recultured ITGA4-containing cells in broth





Last Updated: May 11, 2009 by Fr3P