IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week2/Chemical and Light

Goals for Week 2

1. Transform LacI, TetR, cI + their promoters and GFP. Refer [here].
2. Cut Lac promoter -> GFP (P20), and Tet promoter -> GFP (P15), with Xba and PstI and cut SB3K3 (P5) with Xba and PstI and ligate.
3. Transform cI promoter. (Refer [here].
4. Put constitutive promoters on TetR, LacI, cI.
   1. Put terminators and RBS on TetR, LacI, and cI.
   2. Add promoters.
5. Make primers that have BioBricks prefix and suffix to flank the origin on Duet Vectors so we can take it out.
6. PCR out mtrA and mtrB from Shewie. Also put in with repressors.
7. Test anaerobic growth further with birnesite.

Ongoing Experiments

Sequencing and PCRs

• 6/30: Primers to PCR out the CDF origin of replication (with BioBrick ends) were ordered.
  • 7/1: PCR reaction set up: 45μL PCR supermix, 1μL S1P13 (435ng/μL), 1μL CDF-F primer (20μM), 1μL CDF-R primer (20μM), 2μL water.
    Conditions: 5min @ 94°C → 35x[45s @ 94°C → 45s @ 58°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid
• 6/30: Most of the minipreps of BioBricks that have been done have been prepared to be sent for sequencing. For longer sequences, both the BBsfx and the BBpfx primers were used. For shorter sequences, only the BBsfx primer was used (this primer is longer and therefore gives more leeway for garbled bases at the beginning of a read.)
• 6/30: 5mL LB-Kan culture of P1 is growing (from glycerol stock)- the plasmid can be digested to yield pSB3K3

Re-Transformation of BioBrick Parts in E1 and E3

Miniprepped and made glycerol stocks of Biobrick Parts transformed on Friday.

Name	Registry Name	Description	Origin	Size	Marker	Transformed?	Miniprepped?	Culture in Glycerol Stock?	Verified via sequencing?
P5	pSB3K3	Low-Medium copy vector (w/ death gene)	p15a	2750	Kan	in E3	Yes	Yes (B- done 6/30. A-done 7/1)
P37	BBa_I722007	Constructive expression LacI w/ pTetR promoter			Amp	Failed (liquid cultures didn\'t grow)
P38	BBa_J23114	High constitutive promoter		35	Amp	2007 in E1 (DH5a), 2008 Failed	Yes.	2007 (B- done 6/30. A- done 7/1)
P39	BBa_J23113	Low constitutive promoter		35	Amp	2008 (A grew), 2007 (A grew)	Yes.	2008 (A- done 6/30) 2007 (A-done 6/30)
P40	BBa_B0032	Ribosome Binding Site		13	Amp	2008 (A grew), 2007 (B grew)	Yes.	2007 (B -done 6/30) 2008 (A-done 6/30)
P41	BBa_B0010	Transcriptional Terminator			Amp	Failed (liquid cultures didn\'t grow)
P42	BBa_C0012	LacI coding region			Amp	Failed (liquid cultures didn\'t grow)
P43	BBa_C0040	TetR coding region		660	Amp	2007 (A grew)	Yes	2007 (A-done 6/30)
P44	BBa_C0051	cI lambda		750	Amp	2007 (A grew)	Yes	2007 (A-done 6/30)
P45	BBa_E0240	GFP only w/ RBS & terminator			Amp	2007 (A and B grew)	Yes	2007 (A and B -done 6/30)
P46	BBa_I51020	Base Vector			Amp	A and B grew.	Yes	A and B -done 6/30
P47	BBa_P1003	Kan Resistance Cassette		967	Kan	2007 (A and B grew, but spilled)	Yes	2007 (A and B -done 7/1)
P48	BBa_P1004	Cm Resistance Cassette		769	Cm	2007 (A and B grew, but spilled)	Yes	2007 (A and B -done 7/1)
P49	BBa_P1002	Amp Resistance Cassette		943	Amp	2008 (A grew), 2007 (A and B grew)	Yes	2008 (A-done 6/30) 2007 (A and B -done 6/30)
P50	BBa_P1005	Tet Resistance Cassette		1283	Tet	2007 (A and B grew)	Yes	2007 (A and B- done 7/1)

Restriction Enzyme Digestions - First Round

Vectors (digested with SpeI and PstI)

• RBS (P40, P45)
• cI regulated lambda promoter (P18)
• promoters (P38, P39)

Inserts (digested with XbaI and PstI)

• repressor coding regions (P43, P44)
• GFP under Tet promoter (P15)
• GFP under Lac promoter (P20)
• vector with p15A origin (P1)

Reaction mixture

• 15 μl DNA
• 6.25 μl H₂O
• 2.5 μl 10X NEB buffer (buffer 3 for XP, buffer 2 for SP)
• 0.5 μl 50X BSA
• 0.5 μl each RE

Reactions:

'	P1 (vector)	P15 (insert)	P20 (insert)	P38 (vector)	P39 (vector)	P40 (vector)	P43 (insert)	P44 (insert)	P45 (vector)
DNA	15 uL	15 uL	15 uL	15 uL	15 uL	15 uL	15 uL	15 uL	15 uL
10X Buffer (Volume & #)	2.5 uL of 3	2.5 uL of 3	2.5 uL of 3	2.5 uL of 2	2.5 uL of 2	2.5 uL of 2	2.5 uL of 3	2.5 uL of 3	2.5 uL of 2
50X BSA	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL
Restriction Enzyme 1	0.5 uL XbaI	0.5 uL XbaI	0.5 uL XbaI	0.5 uL SpeI	0.5 uL SpeI	0.5 uL SpeI	0.5 uL XbaI	0.5 uL XbaI	0.5 uL SpeI
Restriction Enzyme 2	0.5 uL PstI	0.5 uL PstI	0.5 uL PstI	0.5 uL PstI	0.5 uL PstI	0.5 uL PstI	0.5 uL PstI	0.5 uL PstI	0.5 uL PstI
Water	6.25 uL	6.25 uL	6.25 uL	6.25 uL	6.25 uL	6.25 uL	6.25 uL	6.25 uL	6.25 uL
Total Volume	25.25 uL	25.25 uL	25.25 uL	25.25 uL	25.25 uL	25.25 uL	25.25 uL	25.25 uL	25.25 uL

Incubate overnight at 37 °C

Gel Extraction and Purification

Gel 1

Well	Sample
1	P1
2	P15
3	P20
4	1 kB Ladder

Gel 2

Well	Sample
1	P40
2	P43
3	P44
4	P45
5	1 kB Ladder

Ligation

We are ligated the repressor coding regions (P43 and P44) to the RBS (P40). We are also going to put P15 and 20 into a p15A vector (P1). After sequencing P18 (cI regulated lambda promoter) we will add it to P45 (RBS with GFP).

We transformed DH5α cells with each of these new plasmids (P40+43, P40+44, P1+15, P1+20).