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Contents 1 Week 6 1.1 Monday 6th August 2007 1.2 Tuesday 7th August 2007 1.3 Wednesday 8th August 2007 1.4 Thursday 9th August 2007 1.5 Friday 10th August 2007

Week 6

Monday 6th August 2007

1. Maia checked the presence and size of (*m*) and (*s*) inserts within the Topo vector by repeating the PCR performed on 02/08. Results confirmed presence of inserts with the correct size. The TOPO vectors containing (*m*) and (*s*) were then sent for sequencing to ensure the inserts did not contain any mistakes. DNA sequenced and the primers used are summarized in the table below.
   

   Label	B1	B3	C5
   Gene	(*m*)	(*s*)	(*m*)
   Colony	17,18,19,26	20	25,24
   Primers	(*m*)_for_1 + Methyl _2	(*s*)_for_1 + Oxy_2	(*m*)_for_1 + (*m*)_rev_1

   (NB - For each 2 x 10µl was sent)
   

2. Christine repeated PCR of the 7 gene operon and 2x 3.5kb gene operons (*a→*d and *d→*g) with the intention to obtain a large amount of PCR product. PCR performed was a repeat of the PCR from 31/07, with annealing temperature of 58°C.

Tuesday 7th August 2007

1. Christine transformed TOP10 cells with TOPO vector containing the 7 gene operon. 4 µl of PCR product was cloned into TOPO vector. Only 2 colonies of (*a→g*) grew, and no (*a→g*)grew. (see Protocol 12)
2. (*d→g*) was amplified again with KOD XL and KOD using the respective programs (see Protocol 9). Primers used: (*d*)_for_1 and (*g*)_rev_1 .Expected size is 3.5 kb.

Wednesday 8th August 2007

1. A digest scheme was designed to allow for the insertion of our biobricks into construction vectors.
   

   Biobricks :	DntR,	(*s*),	(*m*),	XylR,	XylR + Pr,	Pr,	Pu
   Construction Vectors:	3/20G,	4/6B,	4/50

2. (*d→g*) was gell extracted, cloned into TOPO vectors and transformed into TOP10 cells. (see Protocol 12). No (*d→g*) transformations grew.
3. Overnights were set up for (*a→g*) in carbencilln LB.

Thursday 9th August 2007

1. Each biobrick and construction vector was digested with EcoRI and SpeI (Roche)(see Protocol 7)
2. Digest products were resolved on 1% agarose gels and either gel extracted (see Protocol 11) or PCR purified (see Protocol 14)
3. Each biobrick was ligated with three different construction vectors (listed above) using the Quick Ligation Kit (New England Biolabs) (see Protocol 15)
4. Ligation products were transformed into TOP10 cells and grown on LB agar plates supplemented with appropriate antibiotic.
5. Miniprepped overnights of both (*a→g*) colonies according to QIAGEN miniprep kit protocol (see Protocol 5).
6. PCR was carried out on the (*a→g*) minipreps using KOD XL and KOD XL program with annealing temperature of 58°C (see Protocol 9). Primers used were Bbp_*a_for_1 and Bbs_*g_rev_1. No amplicons were observed.
7. Restriction digests were set up for (*a→g*) according to Roche Protocol using Buffer H (see Protocol 7). Expected fragment sizes were not observed.

   (*a→g*) Colony	Enzyme	Fragment Sizes in Orientation 1	Fragment Sizes in Orientation 2
   1 + 2	EcoRI	4582bp, 2622bp, 2997bp, 18bp	6367bp, 2622bp, 18bp, 1212bp
   1 + 2	PstI	7789bp, 486bp, 1451bp, 494bp	4380bp, 486bp, 1451bp, 3902bp

Friday 10th August 2007

1. Results of construction vector transformations: (number of colonies per plate)

   	Construction Vector
   Biobrick	3/20G	4/50	4/6B
   Pu	0	2	3
   Pr	0	V.small	V.small
   XylR	2	3	V.small
   XylR + Pr	0	0	0
   DntR	5	3	3
   (*m*)	12	V.small	8
   (*s*)	31	1	56