Team:TU Delft/15 July 2010 content


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Lab work

Ordered DNA + Solvent Tolerance and Hydrocarbon Sensing

Some of transformations of yesterday containing the different ligations gave colonies (1 a 2 per plate). We picked as many colonies as possible of every plate and performed a colony PCR overnight to check which colonies contained the right insert. At the same time we grow them overnight in 5 mL LB medium containing the appropriate antibiotic.


The results from the Colony PCR were not conclusive yesterday. That is why we decided to isolate the plasmid using Birnboim protocol. We used 3 mL of the bacterial cells to make -80 °C stocks.

The following plasmid concentrations were obtained:

BioBrick Composed of Concentration (ng/μL)
K398203 OprG-B0015 (transformant 6)
K398203 OprG-B0015 (transformant 7)
K398204 AlnA-B0015 (transformant 11)

The plasmids were subsequently digested with an enzyme that characteristic digest for all different plasmids. The plasmids were digested with HindIII. This should yield 3 bands in the lane with the plasmid pSB1T3 containing J04450 (neg control), 1 band in the lane with AlnA-B0015 and 2 bands in the lane with OprG-B0015 on gel.

# Digestion reaction Used Buffer
1 1.5 μg pSB1T3 Buffer 2 + BSA (BioLabs)
2 1 μg OprG-B0015 (transformant 6) Buffer 2 + BSA (BioLabs)
3 1 μg OprG-B0015 (transformant 7) Buffer 2 + BSA (BioLabs)
4 1 μg AlnA-B0015 (transformant 11) Buffer 2 + BSA (BioLabs)

1% Agarose gel of digestion products:

1% Agarose gel of digestion products

Lane description:

# Description Expected Length (bp)
1 SmarLadder marker (5 μL)
2 Undigested pSB1T3 (5 μL + 1 μL loadingbuffer)
3 Digested pSB1T3 (10 μL + 1 μL loadingbuffer) 1972, 1176
4 Undigested OprG-B0015 (transformant 6) (5 μL + 1 μL loadingbuffer)
5 Digested OprG-B0015 (transformant 6) (10 μL + 2 μL loadingbuffer) 1828, 1475, 222
6 Digested OprG-B0015 (transformant 7) (10 μL + 2 μL loadingbuffer) 1828, 1475, 222
7 Undigested AlnA-B0015 (transformant 11) (5 μL + 1 μL loadingbuffer)
8 Digested AlnA-B0015 (transformant 11) (10 μL + 2 μL loadingbuffer) 3511

Characterization of Anderson RBS sequences

Fluorescence measurements attempt #2:

1. Last night's cultures of TOP10 carrying K398500, K398501, K398502, K398503, K398504 and I13401 were measured for biomass (OD600) and diluted to an OD600 of 0.1.

2. Aliquots of 175 uL of the dilutions were pipetted into the wells of a 96-wells plate in three-fold.

3. 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.

Assembly of reference construct:

K081005 yielded no transformants and was thus transformed a second time. Transformants of J23100 and