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From 2010.igem.org

Contents 1 Lab work 1.1 Ordered DNA 1.2 Solvent Tolerance and Hydrocarbon Sensing 1.3 Emulsifier 1.4 Characterization of Anderson RBS sequences

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.

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 [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℃, 200 rpm) were measured for OD600 and diluted 1:100.

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

3. The plate was incubated at 37℃ 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.

Note: surprisingly no overnight growth of the cultures was observed in M9 minimal medium containing glucose.

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.