Team:TU Delft/13 July 2010 content

From 2010.igem.org

(Difference between revisions)
(Characterization of Anderson RBS sequences)
(Characterization of Anderson RBS sequences)
Line 206: Line 206:
4. The fluorescence and OD600 were measured for 16.30 hours with intervals of 0.10 hours by means of the Biotek Synergy plate reader with the excitation filter set at 485nm and the emission filter at 520nm.
4. The fluorescence and OD600 were measured for 16.30 hours with intervals of 0.10 hours by means of the Biotek Synergy plate reader with the excitation filter set at 485nm and the emission filter at 520nm.
-
 
Plasmid purification of overnight cultures carrying K398500, K398501, K398502, K398503, K398504 in pSB1A3 was performed using the Midiprep kit from QIAGEN 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.
Plasmid purification of overnight cultures carrying K398500, K398501, K398502, K398503, K398504 in pSB1A3 was performed using the Midiprep kit from QIAGEN 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.

Revision as of 20:17, 19 July 2010

Contents

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:

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

Solvent Tolerance and Hydrocarbon Sensing

Some BioBricks are in production. Digestion reaction

# Digestion reaction Used Buffer Needed fragment
1 1 μg PhPFDα + EcoRI + SpeI Buffer 2 + BSA (BioLabs) ‘E – PhPFDα – S’
2 1 μg PhPFDβ + EcoRI + SpeI Buffer 2 + BSA (BioLabs) ‘E – PhPFDβ – S’
3 1 μg AlkS + EcoRI + SpeI Buffer 2 + BSA (BioLabs) ‘E – AlkS – S’
4 1 μg PalkB + EcoRI + SpeI Buffer 2 + BSA (BioLabs) ‘E – PalkB – S’
5 1 μg B0032 + XbaI + PstI Buffer 2 + BSA (BioLabs) ‘X – B0032 – P’
6 1 μg B0015 + XbaI + PstI Buffer 2 + BSA (BioLabs) ‘X – B0015 – P’
7 1 μg E0422 + XbaI + PstI Buffer 2 + BSA (BioLabs) ‘X – E0422 – P’
8 3 μg pSB1T3 + EcoRI + PstI Buffer 2 + BSA (BioLabs) ‘E - ---- - 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 gel runned at 100V for 1h
EuroGentec SmartLadder

Lane description

# Description Expected Length (bp)
1 Biorad EZ marker (5 μL)
2 Undigested pSB1T3 (10 μL + 2 μL loadingbuffer)
3 Digested pSB1T3 (10 μL + 2 μL loadingbuffer) 2500
4 Undigested AlnA (10 μL + 2 μL loadingbuffer)
5 Digested AlnA (10 μL + 2 μL loadingbuffer) 1107
6 Undigested OprG (10 μL + 2 μL loadingbuffer)
7 Digested OprG (10 μL + 2 μL loadingbuffer) 744
8 Undigested B0015 (10 μL + 2 μL loadingbuffer)
9 Digested B0015 (10 μL + 2 μL loadingbuffer) 130
10 Undigested R0011 (10 μL + 2 μL loadingbuffer) !
11 Digested R0011 (10 μL + 2 μL loadingbuffer) 55
12 Undigested B0032 (10 μL + 2 μL loadingbuffer)
13 Digested B0032 (10 μL + 2 μL loadingbuffer) 13
14 SmartLadder (5 μL)

! Sample not fully loaded on gel

The band position of the digestion products of AlnA and OprG correspond to their length. The other fragments (R0011, B0032 and B0015) probably run of the gel.

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[1] proposed by the USTC team of 2009:

2. The overnight cultures of E.coli TOP10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 (3 mL LB, 37℃, 200 rpm) were measured for OD600 and diluted 1:100.

3. 100 uL and 300 uL aliquots of the diluted cultures were pipetted into 96-well plates in three-fold.

4. The fluorescence and OD600 were measured for 16.30 hours with intervals of 0.10 hours by means of the Biotek Synergy plate reader with the excitation filter set at 485nm and the emission filter at 520nm.

Plasmid purification of overnight cultures carrying K398500, K398501, K398502, K398503, K398504 in pSB1A3 was performed using the Midiprep kit from QIAGEN 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.