IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week1/Chemical and Light

Chemical

Results from Friday (6/20) Electroporation and Transformation in S1

6/23:

Name	Registry Name	Description	Origin	Marker	Observations
P1a	J23113	Low promoter GFP tester	pMB1	Kan	16 pink colonies of approx 7mm diameter
P2a	J23150	Medium promoter GFP tester	pMB1	Kan	no growth
P3a	J23151	High promoter GFP tester	pMB1	Kan	8 pink colonies of approx 7mm diameter
P12	pETDuet-1	Low copy plasmid	pBR322-derived ColE1	Amp	Lawn
P13	pCDFDuet-1	Low copy plasmid	CloDF13	Sm	41 pink colonies and some small yellow ones that may be E. coli
P14	pCOLADuet-1	Low copy plasmid	ColA	Kan	No growth

Growing cultures of E. coli and S1

6/23:

Picked colonies of E. coli and S1 and grew overnight at 37 and 30 degrees, respectively.

Strain	Plasmid Name	Registry Name	Plate Information	Marker	Colonies Picked
E. coli	P15	A1	6/18 electrocompetent	Cm	2
TOP10	P4	pACYC duet	6/19 restreak electrocomp	Cm	2
TOP10	P7	pSB1A2	6/19 restreak electrocomp big and small	Cm	2 (1 from big, 1 from small)
TOP10	P8	BBa_J04450	6/19 restreak electrocomp big and small	Amp	2 (1 from big, 1 from small)
TOP10	P9	BBa_J04430	6/19 restreak electrocomp big and small	Amp	2 (1 from big, 1 from small)
TOP10	P10	BBa_I715038	6/19 restreak electrocomp big and small	Amp	2 (1 from big, 1 from small)
S1	P4	pACYC duet	6/18 electrocomp	Cm	2
S1	P15	A1	6/19 electrocomp	Cm	2
S1	P13	pCDFDuet-1	6/20 electrocomp	Sm	2
S1	P1a	J23113	6/20 electrocomp	Kan	3
S1	P3a	J23151	6/20 electrocomp	Kan	3

Total number of cultures: 24

6/24:

None of the samples in the LB amp media grew. All the others grew and then were miniprepped or stored in glycerol

Concentrations of DNA from minipreps:

Transformation of P5 and P6 into E3

6/24: P5 and P6 were punched from the book and transformed in E3 (the death resistant strain)

Light

Transformations of P11, P15-20

Colony counts

6/23:

Name	Description	Origin	Size	Marker	Number of Colonies
P11	Light responsive system, dual regulation	pUC19-derived pMB1	4333	Amp/Cm	224
P15	GFP with Tet promoter	ColE1	937	Amp	360
P16	Tet repressible promoter	pMB1	54	Amp	264
P17	Inverter (TetR w/o promoter and Tet promoter)	OriS, P1 lytic, F1	902	Kan	2
P18	lambda promoter (cI regulated)	pMB1	49	Amp	112
P19	RFP with lambda promoter	pMB1	918	Amp	104
P20	GFP (LVA tagged) with Lac promoter	pMB1	1122	Amp, Kan	136

Restreaking

06/23: P16, which has GFP under a constitutive Tet promoter did not appear to fluoresce (none of the fluorescent constructs did), so we restreaked all the of the plates (with the same antibiotic on which they were originally grown). They were left in the 37 degree incubator along with a blank Kan and a blank Amp plate (- controls).

06/24: All of the restreaked plates grew and were streaked to individual colonies. Both the Kan and Amp negative controls (no cells) were blank. However, p15 (GFP under pTet) did not appear to glow.

Overnight cultures

06/23: The above was repeated in 5mL LB liquid cultures instead of plates.

06/24: None of the cultures in LB Amp grew (E1P17 in LB Kan did grow). We recultured the other samples in LB Amp and made a master plate with patches from the same colonies that were cultured. Since others seemed to get similar problems (no growth in liquid culture but growth on plates for Amp), we made fresh LB Amp (from freshly made Amp stock) and repeated the cultures.

Miniprep

06/24: Miniprep of E1P17. The DNA is in the -20 °C iGEM freezer. The DNA concentration is 54.8 ng/μL.

Restriction Digest

06/24: Digest of P17 DNA with Xba1 and Spe1. The entire plasmid is 4425 bp and the short fragment (containing only the biobrick) is 902 bp.

15 μl DNA

6.25 μl water

2.5 μl NEB Buffer 2

0.25 μl 100X BSA

1 μl Xba1

1 μl Spe1

Incubated overnight at 37 °C

Transformations of P22-24

6/24: Transformations occurred in duplicate (TOP10 and DH5α) with Amp selection.