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Laboratory One: Harry, Bogdan, James, Bryant

Laboratory Two: Kate, Mike

Tech Support and Laboratory Three: Parthiv, Chris P., Chris Y.

Laboratory One: Harry, Bogdan, James, Bryant


-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)
		-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)
		-K098010 Plate 3, Well 11N, plasmid pSB3k3 (K)
		-B0015 Plate 1, Well 23L, plasmid pSB1AK3 (AK)
		-E1010 Plate 1, Well 18F, plasmid pSB2K3 (K)


-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


-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.


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


-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


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


-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


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


-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


-Picked colonies from plates and made glycerol stocks


-Transformed gycerol stocks into broth culture


-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.


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


-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


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


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


-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


-Ran a 1% Agarose gel electrophoresis to confirm the products of restriction 
-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


-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


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

Laboratory Two: Kate, Mike

Tech Support and Laboratory Three: Parthiv, Chris P., Chris Y.

Last Updated: May 11, 2009 by Fr3P