Team:TU Delft/16 August 2010 content

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Contents

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 - S’
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 gel run 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 pAlkS cut 1272, 1138, 106 Yes
12 pSB1C3 cut 2035, 1081 Yes (However incomplete digestion, see 3116 bp)
15 BioRad EZ Load Marker Varies Yes

Ligation

Following the digestion the products were ligated for 15 minutes at RT:

# BioBrick Fragment 1 Fragment 2 Destination Vector
1 014C ‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’ ‘X - J61100-rubA4-J61100-rubR-B0015 - P’ ‘E - pSB1C3 - P’
2 020C ‘E - J23100-X-J61100-ladA - S’ ‘X - J61101-ADH - P’ ‘E - pSB1C3 - P’
3 405C ‘E - J23100-X-J61101-PhPFDα - S’ ‘X - J61101-PhPFDβ - P’ ‘E - pSB1C3 - P’
4 327C ‘E - pCaiF - S’ ‘X - B0032 - P’ ‘E - pSB1C3 - P’
5 304C ‘E - pAlkS - S’ ‘X - B0032 - P’ ‘E - pSB1C3 - P’
6 Ligation control None None ‘E - pSB1C3 - P’
7 014C ‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’ ‘X - J61100-rubA4-J61100-rubR-B0015 - P’ ‘E - pSB1C3 - P’ (Pre-shipped linearized plasmid)

The ligations were performed in parallel: one batch using the NEB T4 DNA ligase and buffer, the other batch the Fermentas T4 DNA ligase and buffer. We hope to be able to discern the differences in efficiency between the two (if there are any). 1 uL of the ligation mixes was amplified using the universal primers to screen for pSB1C3 plasmids containing the proper ligated insert. The PCR mix was loaded onto 1% agarose gel:

1% agarose of colony PCR. Gel run at 90V for 50 minutes. Of all samples 5 µL was loaded with 1 µL loadingbuffer. 2.5 µL was loaded of SmartLadder

Lane description

# Description Expected size (bp) OK?
1 Smartladder Varies Yes
2 Ligation mix 014C (NEB) No
3 Ligation mix 020C (NEB) No
4 Ligation mix 405C (NEB) No
5 Ligation mix 327C (NEB) No
6 Ligation mix 304C (NEB) No
7 Ligation mix 014C (Fermentas) No
8 Ligation mix 020C (Fermentas) No
9 Ligation mix 405C (Fermentas) No
10 Ligation mix 327C (Fermentas) No
11 Ligation mix 304C (Fermentas) No
12 Ligation mix 014C with linearized pSB1C3 (NEB) No
13 Ligation mix 014C with linearized pSB1C3 (Fermentas) No
14 Digestion mix of pSB1C3 Yes

Transformation

The ligation products (19 uL) were used to transform 25 uL of commercially competent TOP10 cells (OneShot from Invitrogen) in accordance with the OneShot protocol. In order to test the background the digestion product of pSB1C3 was transformed (digestion control) as well as the ligation controls.

Alkane degradation parallel attempt

We are no longer betting on one horse to ligate the alkane degradation bricks together. So, besides the 3-way ligation mentioned above, we also tried to do a 2 way ligation as well.

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 EcoRI NEBuffer 2 ✓ ‘X - J61100-rubA4-J61100-rubR-B0015 - E’
3 017A EcoRI SpeI AseI NEBuffer 2 ✓ ‘E - J23100-J61100-ladA - S’
4 018A XbaI EcoRI NEBuffer 2 ✓ ‘X - J61101-ADH - E’
5 402A EcoRI SpeI AseI NEBuffer 2 ✓ ‘E - J23100-J61101-PhPFDα - S’
6 403A XbaI EcoRI NEBuffer 2 ✓ ‘X - J61101-PhPFDβ - E’
7 pCaiF EcoRI SpeI PvuII NEBuffer 2 ✓ ‘E - pCaiF - S’
8 pAlkS EcoRI SpeI AseI NEBuffer 2 ✓ ‘E - pAlkS - S’
9 B0032 XbaI EcoRI NEBuffer 2 ✓ ‘X - B0032 - E’

The digestions were executed for 1 hour at 37 degrees.

Ligation

Following the digestion the products were ligated over night at 16 C:

# BioBrick Fragment 1 Vector with Fragment 2
1 014C ‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’ ‘X - J61100-rubA4-J61100-rubR-B0015 - E’
2 020C ‘E - J23100-X-J61100-ladA - S’ ‘X - J61101-ADH - E’
3 405C ‘E - J23100-X-J61101-PhPFDα - S’ ‘X - J61101-PhPFDβ - E’
4 327C ‘E - pCaiF - S’ ‘X - B0032 - E’
5 304C ‘E - pAlkS - S’ ‘X - B0032 - E’
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