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From 2010.igem.org

Contents 1 Alkane degradation 1.1 Digestion 1.2 Ligation 1.3 Transformation

Alkane degradation

Yesterday's transformed competent cells of the 014C, 020C, 405C, 327C and 304C ligation mixes (using the BioBrick assembly method) yielded only red colonies, indicating the lack of positive colonies. It is interesting to note the presence of numerous red colonies on the digest control plate, whereas the ligation control plate (Digested pSB1C3 + ligase buffer + ligase) yielded but 1 red colony (at similar trasnsformation mix concentrations). Based on the information gathered thus far a few hypotheses were made:

Hypothesis I: The ligase buffer inhibits transformation efficiency in some way.

Hypothesis II: The ligation time of 20 mins was too short.

In order to test hypothesis I, the following mixes will be transformed into commercial competent cells:

In order to test hypothesis II, a ligation kinetics assay was performed using E and P digested pSB1C3 as vector and J04450 as insert. The ligations were run for 30, 60, 120 and 240 minutes. Furthermore, a second attempt was made at creating BioBricks 014C, 020C, 405C, 327C and 304C. pSB1C3 was digested additionaly using HindIII to cut the J04450 insert as follows:

#	Fragment 1	Ligase buffer?	Total volume (uL)
1	E-pSB1C3-P (2 uL)	No	20
2	E-pSB1C3-P (2 uL)	Yes (2 uL)	20

#	Sample	Enzyme 1	Enzyme 2	Enzyme 3	Buffer	BSA	Needed fragment
1	pSB1C3	EcoRI	PstI	HindIII	NEBuffer 3	✓	‘E - pSB1C3 - P’

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:

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). To test whether the ligations were succesful, i uL of ligation mix was used as template for a PCR amplification. The results will be known by tomorrow.

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.