Team:WashU/Notebook/Biobricks

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Week of 8/9

8/12

Ordered the following primers to biobrick parts

p18	Forward KanMX4	ATT CTT AGA ATT CGC GGC CGC TTC TAG CCC GCC GCC ACC ATG GGT AAG GAA AAG ACT CAC GTT TCG 
p19	Reverse KanMX4	GCC GCC CTC TGC AGC GGC CGC TAC TAG TAT TAG AAA AAC TCA TCG AGC ATC AAA TGA AAC TG
p20	Forward NatMX4	GTT TCT TCG AAT TCG CGG CCG CTT CTA GCC CGC CGC CAC CAT GGG TAC CAC TCT TGA CGA CAC G
p21	Reverse NatMX4	GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TAG GGG CAG GGC ATG CTC ATG TAG 
p22	Forward Ura3-1	GCACAGAACAATAACCTGCTGGAAACGAAGATAAATCgaagacGATTACTTCGCGTTATGCAGGC
p23	Reverse Ura3-1	GCATCTTCTCAAATATGCTTCCCAGCCTGCTTATCcttctgAAATTCTGCCTCGTGATACGCC
p24	Forward Ura3-2	tactagtagcggccgctgcagTCTTAACCCAACTGCACAGAACAATAACCTGCTGGAAACG
p25	Reverse Ura3-2	ctctagaagcggccgcgaattcTTAGTATTGCTGGCCGCATCTTCTCAAATATGCTTCCCAGCC
p26	Forward His3-1	GAACAGGCCACACAATCGCAAGTGATTAACgaagacGATTACTTCGCGTTATGCAGGC
p27	Reverse His3-1	CCTTGAACGCACTCTCACTACGGTGATGATCActtctgAAATTCTGCCTCGTGATACGCC
p28	Forward His3-2	tactagtagcggccgctgcagAGAGGGAGAAGCAGTAGCAGAACAGGCCACACAATCGCAAG
p29	Reverse His3-2	ctctagaagcggccgcgaattcTTATGGCAACCGCAAGAGCCTTGAACGCACTCTCACTACGG
p30	Biobrick Forward	tgccacctgacgtctaagaa
p31	Biobrick Reverse	attaccgcctttgagtgagc
p32	Forward Ura3 Check	CGG TAA TCT CCG AGC AGA AGG AAG AAC G
p33	Reverse Ura3 Check	CAT TAC GAC CGA GAT TCC CGG GTA ATA ACT G
p34	Forward His3 Check	GAG CAG AAA GCC CTA GTA AAG CGT ATT ACA AAT G
p35	Reverse His3 Check	CTA CAT AAG AAC ACC TTT GGT GGA GGG AAC
p36	Forward SxL	gtttcttcgaattcgcggccgcttctagagcccgccgccaccatgtacg
p37	Reverse SxL	gtttcttcctgcagcggccgctactagtattattataccttgcgctttttcttgggg
p38	Vector internal Sequencing 1 - Forward 1400 start	ACA CCC GTC CTG TGG ATC 
p39	Vector internal Sequencing 2 - Reverse 1523 start	GAAGTGGCGAGCCCGATCTTC
p40	Vector internal Sequencing 3 2301 start	CCACCTCGACCTAACTCGAGTTAC

Week of 8/16

8/19

The following PCr reactions were run and then column Purified.

Number	Product 	Template	Forward Primer 	Reverse Primer 	 Temperature
1	Kan BB 		KanMX4 		p18 		p19 		58 
2	Nat BB 		NatMX4 		p20 		p21 		58 
3	Kan BB 		KanMX4 		p18 		p19 		60 
4	Nat BB 		NatMX4 		p20		p21 		60 
5	Kan BB 		KanMX4 		p18 		p19 		64 
6	Nat BB 		NatMX4 		p20 		p21 		64 

The following digestions of were run.

	K58 	N58 	K60 	N60 	K64 	N64 	P1 
H20	12.5 	12.5 	12.5 	12.5 	12.5 	12.5 	22.5 
Buffer 2	5 	5 	5 	5 	5 	5 	5 
BSA	0.5 	0.5 	0.5 	0.5 	0.5 	0.5 	0.5 
DNA	30 	30 	30 	30 	30 	30 	20 
EcoRI	1 	1 	1 	1 	1 	1 	1 
PstI	1 	1 	1 	1 	1 	1 	1 

The digestions were run for 6 hours at 27 degC and then kept at 4 degC o/n

Made Chloramphenicol Plates at a concentration of 34μg/ml

8/22

Miniprepped the following from overnight cultures made on 8/21/10:

DNA				Concentration (ng/μl)	260/280 	20/230 
Kan BB in pSB1C3 		 37.8			1.92		2.15 
pSB1AT3 with BBa_J04450 insert  102.1			1.92 		2.26 
pSB1C3 with BBa_J04450 insert	 196.8			1.91 		2.31 
	 	 	 

The following PCR reaction was run with an elongation time of 3.5 minutes:

Number	Product 	Template 	Forward Primer 		Reverse Primer 		Annealing Temperature
1	His3 Intermediate Vector	pSB1AT3 w/ insert	 p27		p29 	 58
2	Ura3 intermediate Vector	pSB1AT3 w/ insert	 p23		p25	 58
3	His3 intermediate Vector	pSB1AT3 w/ insert	 p27		p29 	 60
4	Ura3 intermediate Vector	pSB1AT3 w/ insert	 p23		p25 	 60
5	His3 intermediate Vector	pSB1AT3 w/ insert	 p27		p29 	 62
6	Ura3 intermediate Vector	pSB1AT3 w/ insert	 p23		p25 	 62

The following Digestion was run:

	 pSB1C3 w/ insert
H20	 40
DNA	 2.5
NEB-3	 5
BSA	 0.5
EcoRI	 1
PstI	 1


The following Gel was run:

1 1kb Ladder
2 H58 intermediate
3 U58 intermediate
4 H60 intermediate
5 U60 intermediate
6 H62 intermediate
7 U62 intermediate
8 pSB1C3 Digestion product

WashU8-22Gel.jpg

The gel was cut and the products were gel purified using the Sigma Kit

The following PCR reaction was let run over night with an elongation time of 3.5 minutes:

Number	Product 		Template 			Forward Primer 	Reverse Primer 	Annealing Temperature
1	His3 Final Vector	 H58 Intermediate Vector	 p27		p29 	 	58
2	Ura3 Final Vector	 U58 Intermediate Vector	 p23		p25	 	58
3	His3 Final Vector	 H60 Intermediate Vector	 p27		p29 	 	60
4	Ura3 Final Vector	 U60 Intermediate Vector	 p23		p25 		60
5	His3 Final Vector	 H62 Intermediate Vector	 p27		p29 	 	62
6	Ura3 Final Vector	 U62 Intermediate Vector	 p23		p25 	 	62

Week of 8/23

8/23

Nanodropped Over night PCR product:

Number	Label 	Concentration ng/μL 	260/280 	260/230 
1	H58 	10.2 	2.34 	1.22 
2	U58 	6.6 	2.31 	1.13 
3	H60 	10.2 	1.81 	1.14 
4	U60	5.5	2.33	1.16 
5	H62 	7.4 	2.00 	1.18 
6	H62 	0.4 	-0.65 	0.17 
		 	 	 


Preformed the following digestions at 37 degC for 2 hours and heat inactivation at 80 degC for 20 minutes:

	H58 	U58 	H60 	U60 	H62 	 pSB1C3 w/ insert	ADH1 Promoter 	Kan_BB 	Linear pSB1T3	pSB1AT3 w/ insert 
H20	 12.5	12.5 	12.5 	12.5 	12.5 	 40	 40	29.3 	32.5	335.6 
DNA	 30	 30 	 30 	 30 	 30 	 2.5	 2.5	13.2 	 10	 4.9
NEB-2	 -	- 	- 	- 	- 	- 	 5	5 	5 	5 
NEB-3	 5	5 	5 	5 	5 	 5	 -	- 	- 	- 
BSA	 0.5	0.5 	0.5 	0.5 	0.5 	 0.5	0.5 	0.5 	0.5 	0.5 
1μL E1	EcoRI	EcoRI 	EcoRI 	EcoRI 	EcoRI	EcoRI 	 EcoRI	XbaI 	 EcoRI	EcoRI  
1μL E2 	 PstI	PstI  	PstI 	PstI 	PstI 	PstI  	 SpeI	PstI 	 PstI	PstI 
1μL E3	 	 	 	 	 	 	 	 	 	 XbaI
1μL E4	 	 	 	 	 	 	 	 	 	 SpeI

Preformed the following ligations following the Biobrick Manual Protocol

	H58 	U58 	H60 	U60 	H62 	AHD1+K_BB+pSB1T3	ADH1+K_BB+pSB1AT3
H20	 11 	11 	11 	11 	11 	11 	11 
Component 1	H58 	U58 	H60 	U60 	H62 	ADH1 		ADH1 
Component 2	pSB1C3 	pSB1C3  pSB1C3  pSB1C3  pSB1C3 	K_BB 		K_BB 
Component 3	 	 	 	 	 	Linear pSB1T3 	pSB1AT3
10x T4 ligase buffer	2 	2 	2 	2 	2 	2 	2 
T4 DNA Ligase	1 	1 	1 	1 	1 	1 	1 	1
	 	 	 	 	 	 	 

Preformed the following transformations:

1	H58 			Amp 
2	U58 			Amp 
3	H60 			Amp 
4	U60 			Amp 
5	H62 			Amp 
6	N58+pSB1C3		Chlor 
7	N60+pSB1C3 		Chlor 
8	N64+pSB1C3 		Chlor 
9	ADH1+Kan_BB+pSB1T3	Tet 
10	ADH1+Kan_BB+pSB1AT3	Amp 
11	Tet - Control 		Tet 
12	Chlor - Control 	Chlor 

Ran the following Gel:

1	2 	3 	4 	5 	6 	7 	8 
1000bp Ladder	H58 	U58 	H60 	U60 	H62 	U62 	 1000bp

WashU 8-23Gel.jpg

8/24

Biobrick Group

The following transformations were analyzed

1	H58 			Amp 	 No colonies were observed
2	U58 			Amp 	No colonies were observed 
3	H60 			Amp 	No colonies were observed 
4	U60 			Amp 	No colonies were observed 
5	H62 			Amp 	No colonies were observed 
6	N58+pSB1C3		Chlor 	No colonies were observed 
7	N60+pSB1C3 		Chlor 	No colonies were observed 
8	N64+pSB1C3 		Chlor 	No colonies were observed 
9	ADH1+Kan_BB+pSB1T3	Tet 	A Lawn was observed 
10	ADH1+Kan_BB+pSB1AT3	Amp 	~ 80 colonies were observed with ~ 10% showing a red coloring 
11	Tet - Control 		Tet 	 A Lawn was observed
12	Chlor - Control 	Chlor 	 No colonies were observed
	 	 	 

Conclusions: 1. The tetracycline plates did not succesfully select for resistance

The following PCR reactions were run using the sigma supplied PCR kit:

		H64 	U64 	H68 	U68 	C68 
Initial Tm	64 	64 	64 	64 	64 
Second Tm	64 	64 	68 	68 	68 
H20	37.5 	37.5 	37.5 	 37.5	42.5 
PCR Buffer	 5	5 	5 	5 	5 
2.5μL Primer 1	 p26	p22 	p26 	p22 	None 
2.5μL Primer 2	 p27 	p23 	p27 	p23 	None 
pSB1AT3 10ng/μL1 	1 	1 	1 	1 
Nucleotide Mix	1 	1 	1 	1 	1 
Taq Polymerase	0.5 	0.5 	0.5 	0.5 	0.5 
	 	 	 	 	 

These reagents were made following the protocol located at here

The following PCR program was used 1. 94 degC 2:00 2. 94 degC 0:30 3. Initial Tm 1:00 4. 72 degC 3:30 Go to Step 2 x5 5. 94 degC 0:30 6. Second Tm 1:00 7. 72 degC 3:30 Go to Step 5 x 30 8. 68 degC 10:00 9. 4 degC overnight The following digestion was run and Gel Purified:

	 pSB1AT3 x3	 pSB1C3 x3
H20	 37.5		40 
DNA	 5 (102.1ng/μL)	2.5 (197ng/μL)
NEB-3	 5		5 
BSA	 0.5		0.5 
EcoRI	 1		1 
PstI	 1		1 

Digestion Program: 1. 37 degC 2:00:00 2. 80 degC 20:00

WashU 8-24Gel.jpg

1		2 	3 	4 	5 		6 		7 
1kb ladder	pSB1C3 	pSB1C3 	pSB1C3 	pSB1AT3 	pSB1AT3 	pSB1AT3 

The second shortest band representing the linearized plasmid backbone was cut from each column and gel purified. Gel purification was conducted using the Sigma kit, the the elution solution was heated to 65 degrees to accommodate the large Linear DNA fragment. Lanes 2&3 and 4&5 were combined on one column to increase concentration. All elutions were done with 30 μL in order to increase concentration

Nanodrop results:

linear pSB1C3 from lane 1: 9.1 ng/uL linear concentrated pSB1C3 from lanes 2&3: 8.1 ng/ul linear pSB1AT3 from lane 4: 4.1 ng/ul linear concentrated pSB1AT3 from lanes 5&6: 5.5 ng/ul

New LB and Amp Plates were Made

The Kan_BB was submitted to sequencing 1μM dilutions of the biobrick primers were made. The following sequencing reaction was set up for both the forward and reverse primers: 5.3 μL of 37ng/μL Kan_BB DNA 3.2 μL of 1μM plasmid 3.5 μL of H20

Week of 8/9

Week of 8/16

Week of 8/23

Week of 9/6

Week of 9/13

Week of 9/20

Week of 9/27

Week of 10/4