Transformations in Shewie

About 250 ng of DNA added each time.

First Set 8/22

Plate	Colonies	Size	Color
S1 P102 (P108+45) Kan	5 colonies	3mm to 5 mm	Pink
S1 P121+P38 (P124) 1:2.5 Kan A	1 colony	5mm to 7.5mm	Pink
S1 P28 (ColE1) Kan	0 colonies
S1 P121+P38(P124) 1:2.5 Kan B	0 colonies
S1 P121+P39 1:2.5 Dephos Kan A	0 colonies
S1 Topo vector (puc) 2ul Kan X-gal	>50 colonies	1mm	White
S1 Topo vector (puc) 4ul Kan X-gal	>50 colonies	1mm	White
S1 P121+P39 1:2.5 Kan D	0 colonies
S1 (-) ctrl (just cells) Kan	0 colonies
S1 (+) ctrl (E1 p59b) Kan	2 colonies	5mm	Pink

Poor transformation efficiency in the positive control suggests that there may be a problem with our cells. All samples, except for P102, were retransformed the following week with fresh cell cultures.

Second Set (with new cells) 8/27

Plate	Marker	Description
S1 P124 1:2.5 A	Kan	14 2-3mm pink colonies, ~100 0.5mm white colonies
S1 P124 1:2.5 B	Kan	2 pink 2mm colonies, >100 0.5mm white colonies
S1 P125 1:2.5 Dephos A	Kan	2 pink 2mm colonies, >100 0.5mm white colonies
S1 P125 1:2.5 D	Kan	8 2mm pink colonies, ~100 0.5mm white colonies
S1 P28	Kan	~8 1-2mm pink colonies, ~50 0.5mm white colonies
S1 TOPO 2uL	Kan	Lawn of (>200) 0.5mm white colonies
S1 TOPO 4 uL (w/ Xgal)	Kan	Lawn of (>200) 0.5mm white colonies
S1 P59b (+) ctrl	Kan	~50 2mm pink colonies, ~50 0.5mm white colonies
S1 (-) ctrl	Kan	50-100 0.5mm white colonies

It is unclear if the puc origin works because in both sets of transformations, the colonies that grow don't resemble S1 at all. This is at least the 2nd or 3rd instance wherein ColE1 has worked in S1 in P28. The better efficiency of the second set suggests the frozen stocks of S1 competent cells may not be as viable anymore, but we should test this further if we have time before throwing them away-- Shewie take forever to grow to log phase!

Getting Thermoinducible and Lac Inducible GFP into pSC101

Digests of Inducible Systems and pSC101 8/27

P102 needs to be retransformed so it was not included in this set.

Gel of Digest 8/27

	1% agarose
	Lane	Sample
	1	1 kb + DNA Ladder
	2	P124 1:2.5 A
	3	P124 1:2.5 B
	4	P125 1:2.5 Dephos A
	5	P125 1:2.5 D
	6	mtrB TOPO 0.5 uL B
	7	mtrB TOPO 2 uL B
	8	mtrB TOPO 4-1
	9	mtrB TOPO 4-10

mtrB TOPO bands were cut (2.2 kB) but P124-5 did not cut properly, suggesting that there was an extra cut site that was cutting the insert into two pieces.

Single-cut Digest of P124-5 8/27

	1.2% E-gel visualized using EtBr/UV
	Lane	Sample
	1	1 kb + DNA Ladder
	2	P124 1:2.5 A with XbaI
	3	P124 1:2.5 B with XbaI
	4	P125 1:2.5 Dephos A with XbaI
	5	P125 1:2.5 D with XbaI
	7	P124 1:2.5 A with PstI
	8	P124 1:2.5 B with PstI
	9	P125 1:2.5 Dephos A with PstI
	10	P125 1:2.5 D with PstI
	11	1 kb + DNA Ladder

IMPORTANT NOTE Further inspection of the sequence revealed that there was an extra PstI site after the cI repressor coding region due to an error that was made while making the primers that resulted in the suffix being backward and the cut sites on the part to read EXPS. Attempts to mutate out the extra site and basepairs will begin next week once primers are reviewed and ordered-- might take a while just to get them.

Due to the extra PstI site, all future ligations involving the thermoinducible system cut with PstI will need to be held off (ie. getting into the pSC101 vector, and adding on mtrB or mtrA) until the issue is resolved either with site directed mutagenesis or by redesigning primers for cI and starting over?

Re-Transformations of Various Plasmids

Scheduled for next week.

Getting mtrB onto thermo and lac inducible systems

Ligating mtrB TOPO with RBS

Grew up more mtrB TOPO for next week. Will need to grow up more P40 and P97 next week then, too.

PCR of mtrB with RBS + mtrB primers