Latest revision as of 19:57, 5 August 2010

Lab work

Ordered DNA

We have now stock of the ordered DNA, to make a real BioBrick of this DNA we are going to ligated it into the iGEM plasmid backbone SB1C3. First we digested the plasmids:

#	Sample	Enzyme 1	Enzyme 2	Buffer	BSA	Needed fragment
1	1 μg alkB2	EcoRI	PstI	3 (BioLabs)	✗	‘E–alkB2–P’
2	1 μg rubA3	EcoRI	PstI	3 (BioLabs)	✗	‘E–rubA3–P’
3	1 μg ladA	EcoRI	PstI	3 (BioLabs)	✗	‘E–ladA–P’
4	1 μg ADH	EcoRI	PstI	3 (BioLabs)	✗	‘E–ADH–P’
5	1 μg AlnA	EcoRI	PstI	3 (BioLabs)	✗	‘E–AlnA–P’
6	1 μg OprG	EcoRI	PstI	3 (BioLabs)	✗	‘E–OprG–P’
7	1 μg AlkS	EcoRI	PstI	3 (BioLabs)	✗	‘E–AlkS–P’
8	1 μg PalkB	EcoRI	PstI	3 (BioLabs)	✗	‘E–PalkB–P’
9	1 μg PalkS12	EcoRI	PstI	3 (BioLabs)	✗	‘E–PalkS12-P’
10	1 μg PhPFDα	EcoRI	PstI	3 (BioLabs)	✗	‘E–PhPFDα–P’
11	1 μg PhPFDβ	EcoRI	PstI	3 (BioLabs)	✗	‘E–PhPFDβ–P’
12	3 μg pSB1C3	EcoRI	PstI	3 (BioLabs)	✗	‘E–linear pSB1C3–P’

Solvent Tolerance and Hydrocarbon Sensing

Some BioBricks are in production. Digestion reaction

#	Sample	Enzyme 1	Enzyme 2	Buffer	BSA	Needed fragment
1	1 μg PhPFDα	EcoRI	SpeI	2 (BioLabs)	✓	‘E–PhPFDα–S’
2	1 μg PhPFDβ	EcoRI	SpeI	2 (BioLabs)	✓	‘E–PhPFDβ–S’
3	1 μg AlkS	EcoRI	SpeI	2 (BioLabs)	✓	‘E–AlkS–S’
4	1 μg PalkB	EcoRI	SpeI	2 (BioLabs)	✓	‘E–PalkB–S’
5	1 μg B0032	XbaI	PstI	2 (BioLabs)	✓	‘X–B0032–P’
6	1 μg B0015	XbaI	PstI	2 (BioLabs)	✓	‘X–B0015–P’
7	1 μg E0422	XbaI	PstI	2 (BioLabs)	✓	‘X–E0422–P’
8	3 μg pSB1T3	EcoRI	SpeI	2 (BioLabs)	✓	‘E-linear pSB1T3-P’

Emulsifier

Today the digestion products from yesterday were run on gel to see whether the plasmids were cut in the right way.

1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 10 μL + 2 μL loadingbuffer was loaded and 5 μL was loaded of marker

Lane description:

#	Description	Expected Length (bp)	Status	Remarks
1	Biorad EZ marker	n/a	n/a
2	Undigested pSB1T3	3507	✓
3	pSB1T3 + EcoRI + PstI	1085, 2422	✓
4	Undigested AlnA	3469	✓
5	AlnA + EcoRI + SpeI	1071, 2398	✓
6	Undigested OprG	3122	✓
7	OprG + EcoRI + SpeI	708, 2414	✓
8	Undigested B0015	3318	✓
9	B0015 + XbaI + PstI	155, 3163	✓	fragment probably run of the gel
10	Undigested R0011	2134	✓	Sample not fully loaded on gel
11	R0011 = EcoRI + SpeI	78, 2056	?	fragment probably run of the gel
12	Undigested B0032	2092	✓
13	B0032 + XbaI + PstI	39, 2053	?	fragment probably run of the gel
14	SmartLadder	n/a	n/a

The ligation products that were incubated over night were transformed to Top10 competent cells according to the protocol.

Characterization of Anderson RBS sequences

The first attempt at measuring fluorescence was made in adherence with the [http://partsregistry.org/Part:BBa_K176011 protocol] proposed by the USTC team of 2009:

1. The overnight cultures of E.coli Top10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 (LB, 37 ℃, 160 rpm) were measured for OD600 (see table below) and diluted 1:100.

#	OD600
K398500	1.443
K398501	1.410
K398502	1.448
K398503	1.414
K398504	1.552
I13401	1.451

2. 100 μL and 300 μL aliquots of the diluted cultures were pipetted into 96-well plates in three-fold.

3. The plate was incubated at 37 ℃ while shaking for 3 hours.

4. The fluorescence and OD600 were measured for 16.30 hours with intervals 10 minutes by means of the Biotek Synergy plate reader with the excitation filter set at 485nm and the emission filter at 520nm. LB medium was used as blank reference.

Note: surprisingly no over night growth of the cultures was observed in M9 minimal medium containing glucose. This a larger volume of inoculate (in LB) was used for a second stab at over night growth in M9.

Plasmid purification of overnight cultures carrying K398500, K398501, K398502, K398503, K398504 in pSB1A3 was performed using the Qiagen Midi-prep plasmid isolation kit yielding sufficient plasmid DNA. These BioBricks can in future be placed under control of a low to medium copy number plasmid for comparison with high copy-number plasmid expression.

@@ Line 190: / Line 190: @@
 [[Image:TU_Delft_2010-07-13_P_Digestion.png|thumb|left|600px|1 % agarose of digestion check. Gel runned 1 hour at 100 V. Of all samples 10 μL + 2 μL loadingbuffer was loaded and 5 μL was loaded of marker]]
-[[Image:TU_Delft_SmartLadder.jpg|thumb|right|EuroGentec SmartLadder]]
 Lane description:

Team:TU Delft/13 July 2010 content

From 2010.igem.org