Team:TU Delft/16 August 2010 content

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Alkane degradation

Unfortunately last week's attempts hadn't yielded any BioBricks making use of the 2-way ligation method. We thus decided to take another stab at the 3-way ligation procedure, according to the BioBrick Assembly Manual of Ginkgo Bioworks. The BioBrick numbers we tried to construct were: 014C, 020C, 405C, 327C and 304C.

Digestion

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 011A EcoRI SpeI AseI NEBuffer 2 ‘E - J23100-J61100-alkB2-J61100-rubA3 - S’
2 013K XbaI PstI HindIII NEBuffer 2 ‘X - J61100-rubA4-J61100-rubR-B0015 - P’
3 017A EcoRI SpeI AseI NEBuffer 2 ‘E - J23100-J61100-ladA - S’
4 018A XbaI PstI AseI NEBuffer 2 ‘X - J61101-ADH - P’
5 402A EcoRI SpeI AseI NEBuffer 2 ‘E - J23100-J61101-PhPFDα - S’
6 403A XbaI PstI AseI NEBuffer 2 ‘X - J61101-PhPFDβ - P’
7 pCaiF EcoRI SpeI AseI NEBuffer 2 ‘E - pCaiF - S’
8 B0032 XbaI PstI AseI NEBuffer 2 ‘X - B0032 - P’
9 B0032 XbaI PstI AseI NEBuffer 2 ‘X - B0032 - P’
10 pAlkS EcoRI SpeI AseI NEBuffer 2 ‘E - pAlkS - P’
11 pSB1C3 EcoRI PstI None NEBuffer 2 ‘E - pSB1C3 - P’

The digestions were executed for 15 minutes at 37 degrees, and checked on 1% agarose gel:

1% agarose of colony PCR. Gel runned at 100V for 1 hour. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker

Lane description

# Description Expected size (bp) OK?
1 Smartladder Varies Yes
2 011A cut 1523, 1199, 857 Yes
3 013K cut 1825, 1592, 1342, 43 Yes
4 017A cut 1410, 1199, 857 Yes
5 018A cut 1185, 872, 793, 43 Yes
6 402A cut 1199, 857, 543 Yes
7 403A cut 1185, 872, 397, 43 Yes
8 pCaiF in pANY cut 1277, 1143, 74 Yes
9 B0032 in pSB1A2 cut 1185, 872, 35 Yes
10 B0032 in pSB1A2 cut 1185, 872, 35 Yes
11 pSB1C3 cut 2035, 1081 Yes (However incomplete digestion, see 3116 bp)
15 BioRad EZ Load Marker Varies Yes