Team:Freiburg/Notebook/1 August

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Contents 1 green light receptor 1.1 Qickchange PCR of CcaS 2 blue light receptor 2.1 PCR 3 red light receptor 3.1 3A-assembly with pcyA and the terminator BBa_1006 3.1.1 1.Digestion: 3.1.2 2.Ligation 3.1.3 3.Transformation 4 Lysis cassette 4.1 Phage Lysis Cassette (K124017) + RBS (B0034) 4.1.1 Digestion of 3A Assembly 4.1.2 Ligation 4.1.3 Transformation 5 Precipitator 5.1 PCR 5.2 3A-assembly 5.2.1 1.Digestion

green light receptor

Qickchange PCR of CcaS

Investigators:Julia
repeating experiment from___, because we had no positive transformation, probably chosen wrong annealing temperature before.

blue light receptor

PCR

Investigators: Sophie

As our last PCRs of the LovTAP with the Gibson overhangs didn't work well, we now try a PCR with primers without overhangs for Gibson assembly.

Name: Sophie	Date: 01.08.11
Continue from Experiment: PCR (Date): 27.07.11 (Name): Sandra, Sophie
Project Name: Blue Light

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H20	Name
10µl	5x Phusion Buffer	of Primer
2.5µl	Primer fw	P35
2.5µl	Primer dw	P36
1µl	dNTPs	of Template DNA
1µl	DNA-Template	M35 (LovTAP)
0.5 µl	Phusion (add in the end)

What program do you use?

LovTAP ohne Ueberhaenge

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

How did you label the PCR-Product, where is it stored and what do you do next?

Labelled: M35 short; stored in Gibson Stuff box.

red light receptor

3A-assembly with pcyA and the terminator BBa_1006

Investigators:Julia

1.Digestion:

terminator (S21a), 26µl of the miniprep+ 12,1 µl H2O, cut with XbaI&PstI
pcyA (part BBa_I15009), 3 µl of miniprep + 35µl H2O, cut with EcoRI&SpeI
Vector-backbone (10 µl) pSB1T3, cut with EcoRI,PstI & DpnI

to each reation 5µl BSA(10x) and 5µl NEB buffer4 were added, digested at 37°C for two hours, inactivated at 80°C for 20min.

2.Ligation

2µl of digested pcyA,terminator and vector were added to 11µl H2O,2µl ligase buffer and 1µl T4 ligase.
Incubated at room temperature for 35 min, inactivated at 80°C for 20 min.

3.Transformation

Lysis cassette

Phage Lysis Cassette (K124017) + RBS (B0034)

Investigators: Theo

Digestion of 3A Assembly

Name: Theo	Date: 01.08.2011
Continue from Experiment : -
Project Name: Phage Lysis Cassette (K124017) + RBS (B0034)

Procedure

1. add H₂O (38μl-DNA )
2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
6. heat for 1-2 hours 37°C (6 hours if time)
7. heat for 20 minutes 80°C (inactivation of enzymes)
8. keep at 4°C if you cannot continue

Restriction enzymes you need to cut the vector, insert1 and insert 2:

Components	Vector (μl)		Insert1 -B0034- and 2 -K124017-(μl)
DNA (500ng)		10		9	8
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl)		EcoRI + DpnI		EcoRI	XbaI
Enzyme 2 (1μl)		PstI		SpeI	PstI
H2O (38 μl- DNA)		27		29	30
In total 50 μl

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.

A RBS (ribosome binding site) between the lamba cI regulated Promotor (R0051 of K098995) and the holin ATG of K124017 is absent. So a 3A assembly had to be performed in order to get a functioning Lysis cassette. Resistance of RBS (B0034)=Cm and of Phage Lysis Cassette (K124017)=Amp, so the Vector is Tet (pSB1T3)

Ligation

Name: Theo	Date: 01.08.2011
Continue from: 01.08.2011 Phage Lysis Cassette (K124017) + RBS (B0034) Digestion
Project Name: Phage Lysis Cassette (K124017) + RBS (B0034) Ligation

Procedure

PCR tube:

total volume 20 μl

1. add H₂O (17 μl -X-Y-Z)
2. add 2 μl Ligase Buffer 10x
3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
5. Add 1 μl T4-DNA Ligase
6. Incubate 10-30 min at room temperature
7. heat for 20 minutes at 80°C
8. store at -20°C or directly proceed to transformation

	Name of part	Ratio Insert:Vector = 3:1 or 1:1	Volume (μl)
X insert 1	B0034	3:1 (1*3)	3
Y insert 2	K124017	3:1 (2*3)	6
Z vector	pSB1T3	1:3 (1)	1
H2O			7

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.

Ligation step of 3A assembly. How to calculate ratios --> e.g. for K124017 Length of pSB1T3= ca 2200bp Length of K124017+Vector= ca 4500bp 4500/2200= ca 2 So 2*3 µl (since 3:1 is needed) = 6 µl

Transformation

Name: Theo	Date: 01.08.2011
Continue from : 01.08.2011 Phage Lysis Cassette (K124017) + RBS (B0034) Ligation
Project Name: Phage Lysis Cassette (K124017) + RBS (B0034)

Procedure

1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
2. thaw cells on ice 20 minutes
3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
4. Incubate for 30 minutes on ice
5. Heat at 42°C for 60 sec
6. Incubate on ice for 5 minutes
7. Add 200 μl LB Broth
8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
9. Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

Documentation:

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.

New Composite Part in pSB1T3 Vector with modified K124017 containing a strong RBS in front of first ATG RESULT: No colonies!!! :( Probably because of the Tet vector, since other members of the group also had problems. We are trying it again with the pSB1C3 Vector

Precipitator

PCR

Name: Sophie	Date: 01.08.11
Continue from Experiment: replay of PCR 19.07.11 (Date) 19.07.11 (Name) Ruediger
Project Name: GFP Pbd

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H20	Name
10µl	5x Phusion Buffer	of Primer
2.5µl	Primer fw	P28
2.5µl	Primer dw	P18 / P19/ P20
1µl	dNTPs	of Template DNA
1µl	DNA-Template	S 14 (GFP)
0.5 µl	Phusion (add in the end)

What program do you use?

Ruediger PCR (modified by Tobi and me. First 10 cycles with 55°C, next cycles with 62°C annealing temperature.

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

How did you label the PCR-Product, where is it stored and what do you do next?

3A-assembly

1.Digestion

Investigators: Theo

Digestion of PCR product.

• S39
• S43

were digested with EcoRI and SpeI

• P18
• P19
• P20

were digested with XbaI and PstI.