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From 2010.igem.org

Contents 1 Lab work 1.1 Ordered DNA 1.2 Alkane degradation 1.3 Salt Tolerance 1.4 Emulsifier 1.5 Characterisation of Anderson RBS sequences 1.5.1 Assembly of reference construct 1.5.1.1 Method 1 1.5.1.2 Method 4 1.6 Solvent Tolerance and Hydrocarbon Sensing 1.6.1 Characteristic digestion of isolated plasmids

Lab work

Ordered DNA

The transformations of 19 July containing the different ligations gave colonies (~5 per plate). We picked 5 colonies per plate and performed a colony PCR.

Alkane degradation

Unfortunately there were no transformants on yesterday's plates. This is most likely due to the fact that the ligation didn't work with the small pieces of DNA of the RBSs. Next plan is to cut open the RBS plasmid without removing the RBS (with SpeI and PstI) and insert the gene in this plasmid. We will try this tomorrow.

Salt Tolerance

The overnight ligation was transformed using the standard method and grown up overnight at 37 degrees C.

Emulsifier

There were small colonies on the plates. Pieter picked seven and performed colony PCR on them.

Lane Description

#	Description	Expected lenght (bp)	Primer	Status
M1	EZ Ladder	n/a	n/a	n/a
1	B0032 (control)	220	G00100 + G00101	✓
2	Transformant #1 of ligation mix R0011-B0032	300	G00100 + G00101	✗
3	Transformant #2 of ligation mix R0011-B0032	300	G00100 + G00101	✗
4	Transformant #3 of ligation mix R0011-B0032	300	G00100 + G00101	✗
5	Transformant #4 of ligation mix R0011-B0032	300	G00100 + G00101	✗
6	Transformant #5 of ligation mix R0011-B0032	300	G00100 + G00101	✗
7	Transformant #6 of ligation mix R0011-B0032	300	G00100 + G00101	✗
8	Transformant #7 of ligation mix R0011-B0032	300	G00100 + G00101	✗

Unfortunately all other lanes were empty or the same as the negative control. So from this gel we conclude that the ligation has failed.

Characterisation of Anderson RBS sequences

Assembly of reference construct

Method 1

Hypothesis II The transformants that were replated on kanamycin plates produced colonies, indicating that hypothesis II was invalid.

Method 4

The obtained colonies on ampicillin plates were screened for GFP fluorescence, those that fluoresced were inoculated in 5 mL of LB medium for over night growth.

Solvent Tolerance and Hydrocarbon Sensing

Characteristic digestion of isolated plasmids

To determine whether the plasmids are correct. Characteristic cuts were made using the restriction enzyme PvuI.

#	Sample	Enzyme 1	Buffer	Needed fragments
1	1450 μg K398328T	PvuII	Buffer II	252, 1647, 69, 36, 3246
2	378 μg K398407T	PvuII	Buffer II	2552
3	716.5 μg Control (pSB1T3, B0015: this does not ligate)	PvuII (this enzyme does not work here)	Buffer II	172, 2463
4	245.4 μg K398300C	PvuII	Buffer II	654, 252, 1647, 69, 36, 2004
5	1481.4 μg K398000C	PvuII	Buffer II	495, 2901
6	1198.5 μg K398002C	PvuII	Buffer II	2243
7	42.3 μg K398002C	PvuII	Buffer II	3120
8	1612.8 μg K398200C	PvuII	Buffer II	558, 135, 177, 1887
9	1633.3 μg K398201C	PvuII	Buffer II	2529
10	938.6 μg K398400C	PvuII	Buffer II

Results of the digestion on 1% agarose gel

Title figure: 1% Agarose of digestion check gel runned at 100V for 1 hour loaded 5 μL of marker and 10 μL sample + 2 μL loadingbuffer

Lane description:

#	Description	Expected Length (bp)	Status	Remarks
1	marker	n/a	n/a	n/a
2	Digested K398328T	252, 1647, 69, 36, 3246	✗
3	Digested K398407T	2552	✗
4	pSB1T3 with B0015 (control)	172, 2463	✗	The upper band is most likely circular pSB1T3. There's a strange band around 600 bp.
5	Digested K398000C	654, 252, 1647, 69, 36, 2004	✗
6	Digested K398000C	495, 2901	?	There's no band of 495 bp
7	Digested K398002C	2243	✓
8	Digested K398200C	3120	✗
9	Digested K398201C	558, 135, 177, 1887	✓
10	Digested K398400C	2529	✗
11	pSB1T3 (control)	2463	?	A bright band around 2500 bp was shown. There are also a lot of unexplainable bands visible.