Team:TU Delft/2 August 2010 content

From 2010.igem.org

Contents

Lab work

Alkane Sensing, Solvent Tolerance and Salt Tolerance

by Pieter

The plates containing yesterday's ligations contained colonies, to check whether they really contain the desired BioBrick a colony PCR was done, and the used colonies were grown in liquid LB medium over night. The results from the PCR were analysed on a 1% agarose gel.

TU Delft Pi2 Colony PCR part 1.png

  • BBa_K398305 = Alks -> E0240
  • BBa_K398101 = bbc1 -> J61101
  • BBa_K398402 = PhPFDalpha -> J61101
  • BBa_K398403 = PhPFDbeta -> J61101

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
M SmartLadder n/a n/a n/a
1 BBa_K398305 3772 G00101 + G00100 X
2 BBa_K398305 3772 G00101 + G00100 X
3 BBa_K398305 3772 G00101 + G00100 X
4 BBa_K398305 3772 G00101 + G00100 X
5 BBa_K398305 3772 G00101 + G00100 X
6 BBa_K398101 1007 G00101 + G00100 X
7 BBa_K398101 1007 G00101 + G00100 X
8 BBa_K398101 1007 G00101 + G00100 X
m EZ Ladder n/a n/a n/a
M SmartLadder n/a n/a n/a
1 BBa_K398101 1007 G00101 + G00100 X
2 BBa_K398101 1007 G00101 + G00100 X
3 BBa_K398402 836 G00101 + G00100 X
4 BBa_K398402 836 G00101 + G00100 X
5 BBa_K398402 836 G00101 + G00100 X
6 BBa_K398402 836 G00101 + G00100 X
7 BBa_K398402 836 G00101 + G00100 X
8 BBa_K398403 734 G00101 + G00100 X
m EZ Ladder n/a n/a n/a

TU Delft Pi2 Colony PCR part 2.png

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
M SmartLadder n/a n/a n/a
1 - 7 Eva's samples n/a n/a n/a
m EZ tLadder n/a n/a n/a
1 BBa_K398403 734 G00101 + G00100 X
2 BBa_K398403 734 G00101 + G00100 X
3 BBa_K398403 734 G00101 + G00100 X
4 BBa_K398403 734 G00101 + G00100 X
M SmartLadder n/a n/a n/a

Emulsifier

When going over the plates in the fridge we noticed a few white colonies on plates from 13-07-'10. Previously we thought there were only red colonies. So we decided to do a colony PCR on all 12 white colonies.

TU Delft Pi3 Colony PCR.jpg

  • BBa_K398202 = R0011 + B0032

Lane Description:

# Description Expected Length (bp) Primers Status Remarks
M SmartLadder n/a n/a n/a
1 BBa_K398202 392 G00101 + G00100
2 BBa_K398202 392 G00101 + G00100
3 BBa_K398202 392 G00101 + G00100
4 BBa_K398202 392 G00101 + G00100
5 BBa_K398202 392 G00101 + G00100
6 BBa_K398202 392 G00101 + G00100
7 BBa_K398202 392 G00101 + G00100
8 BBa_K398202 392 G00101 + G00100
9 BBa_K398202 392 G00101 + G00100 Isolated plasmid
10 BBa_K398202 392 G00101 + G00100
11 BBa_K398202 392 G00101 + G00100
12 BBa_K398202 392 G00101 + G00100 Isolated plasmid

None of the bands is truly positive. Still number 4,5 and 9 could be positive. Or due to the GC content the bands could be shifted a little bit upwards, so that maybe even sample 12 could be positive.

Just to be sure, we isolated the plasmids and send them to BaseClear for sequencing. The results will be in by Thursday.

Alkane degradation

For the next step in BioBrick formation a digestion was done:

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 1 μg 007T EcoRI SpeI BamH1 2 (Biolabs) ✓ ‘E – J61100-AlkB2 – S’
2 1 μg 008A EcoRI XbaI 2 (Biolabs) ✓ ‘E – J61100-rubA3 – X’
3 1 μg 010A EcoRI SpeI AseI 2 (Biolabs) ✓ ‘E – J61100-rubR – S’
4 2 μg B0015 EcoRI XbaI 2 (Biolabs) ✓ ‘E – B0015 – pSB1AK3 – X’
5 1 μg 017A EcoRI SpeI AseI 2 (Biolabs) ✓ ‘E – J61100-ladA – S’
6 1 μg 018A XbaI PstI AseI 2 (Biolabs) ✓ ‘X – J61101-ADH – P’
7 1 μg 019A EcoRI SpeI AseI 2 (Biolabs) ✓ ‘E – J61107-ALDH – S’
8 1 μg pSB1K3 EcoRI PstI 2 (Biolabs) ✓ ‘E – pSB1K3 – P’

Results of the digestion on 1% agarose gel

1% Agarose of digestion check

The gel was runned at 100V for 1 hour, loaded 5 μL of marker and 5 μL sample + 1 μL loadingbuffer

Lane description:

# Description Expected Length (bp) Status
1 Smartladder
2 ‘E – J61100-AlkB2 – S’ (007T) ✗
3 Undigested 007T ✗
4 ‘E – J61100-rubA3 – X’ (008A) ✓
5 Undigested 008A ✓
6 ‘E – J61100-rubR – S’ (010A) ✓
7 Undigested 010A ✓
8 ‘E – B0015 – pSB1AK3 – X’ ✓
9 pSB1AK3-B0015 ✓
10 ‘E – J61100-ladA – S’ (017A) ✓
11 Undigested 017A ✓
12 ‘X – J61101-ADH – P’ (018A) ✓
13 Undigested 018A ✓
14 ‘E – J61107-ALDH – S’ (019A) ✓
15 Undigested (019A) ✓
16 ‘E – pSB1K3 – P’ ✓
17 Smartladder
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