Team:TU Delft/19 July 2010 content

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'''Digestion'''
'''Digestion'''
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The PCR product of promotor R0011 was cut with EcoRI and SpeI and the RBS containing plasmid has been cut open with EcoRI and Xbal at 37 C.
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The PCR product of promotor R0011 was cut with EcoRI and SpeI and the RBS containing plasmid has been cut open with EcoRI and Xbal at 37 C. This deviates from the standard biobrick assembly, thus is not completely as described in our [[Team:TU_Delft/protocols/restriction_enzyme_digestion|digestion protocol]].
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Revision as of 14:50, 19 July 2010

Lab work

Alkane degradation

Biobricks in production:

Digestion reactions:

# Digestion reaction Used Buffer Needed fragment
1 2 μg J61100 + EcoRI + SpeI NEBuffer 2 ‘E – J61100 – S’
2 2 μg J61100 + EcoRI + SpeI NEBuffer 2 ‘E – J61100 – S’
3 1 μg rubR + EcoRI + SpeI NEBuffer 2 ‘E – rubR– S’
4 1 μg J61101 + EcoRI + SpeI NEBuffer 2 ‘E – J61101– S’
5 1 μg J61107 + EcoRI + SpeI NEBuffer 2 ‘E – J61107– S’
6 1μg alkB2 + Xbal + PstI NEBuffer 2 ‘X – alkB2 – P’
7 1 μg rubA3 + Xbal + PstI NEBuffer 2 ‘X – rubA3 – P’
8 1 μg rubA4 + Xbal + PstI NEBuffer 2 ‘X – rubA4 – P’
9 1 μg B0015 + Xbal + PstI NEBuffer 2 ‘X – B0015 – P’
10 1 μg ladA + Xbal + PstI NEBuffer 2 ‘X – ladA – P’
11 1 μg ADH + Xbal + PstI NEBuffer 2 ‘X – ADH – P’
12 1 μg ALDH + Xbal + PstI NEBuffer 2 ‘X – ALDH – P’
13 3 μg pSB1T3 + EcoRI + PstI NEBuffer 2 ‘E – pSB1T3(lin) – P’

Emulsifier

Since last weeks attempt to construct the inducible protomer R0011 with RBS B0032 failed, we decided to take a different approach. Instead of doing the standard 2 parts assembly, we amplify the promoter by PCR digest it and ligate in the plasmid that contains the RBS which has only be cut open at the left side. By doing so we do not risk losing the super small DNA fragments like the RBS. The problem is that we cannot use antibiotics selection. So we need to determine that by Colony PCR and sequencing.

PCR Amplification

[http://partsregistry.org/Part:BBa_R0011 R0011] was amplified with the universal primers. The product was put on gel:

1% agarose gel

Lane description:

# Description Amount
1 BioRad EZ Ladder 5 ul
2 PCR Product 10 ul + 2 ul LB

The PCR band is about 300 bps long. R0011 itself is just 55 bp, but the flanking primer regions are about 100 bp each.

Digestion

The PCR product of promotor R0011 was cut with EcoRI and SpeI and the RBS containing plasmid has been cut open with EcoRI and Xbal at 37 C. This deviates from the standard biobrick assembly, thus is not completely as described in our digestion protocol.

# Digestion reaction Used Buffer Needed fragment
1 1 μg B0032 + EcoRI + Xbal: Buffer 2 ‘X – B0032 - Plasmid backbone – E’
2 30 μl PCR product of R0011 + EcoRI + SpeI Buffer 2 ‘E - R0011 - S’

Ligation

The digestion products were ligated over night:

# Ligation reaction
1 5 μL Cut plasmid with B0032 + 10 μL PCR Product of R0011

Hopefully this will lead to E - X - R0011 - B0032 - S - P in the B0032 plasmid backbone with Amp resistance marker.

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