From 2008.igem.org

1 Goals for Week 2
2 Ongoing Experiments
- 2.1 Sequencing and PCRs
3 The Plan
- 3.1 Scoreboard: Keeping track of what we have and don't have
4 Transformations from the Registry
5 First Round
6 Second Round
- 6.1 Digestions (7/3)
7 Transformation of Parts into S1
- 7.1 Transformation of Lac/Tet + GFP into S1(7/3)

Goals for Week 2

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

The Plan

Goal: High/ Low Constitutive Promoter + RBS + Repressor Coding Region for cI Lambda/ TetR/ LacI + Terminator(s) + pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori

Scoreboard: Keeping track of what we have and don't have

RBS + Repressor Coding Region

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P56	RBS + TetR (P40+P43)	Yes (7/1)	Yes (7/2)	Yes (7/2)	Yes, in E1 (7/2)
P57	RBS + cI lambda (P40+P44)	Yes (7/1)	Yes (7/2)	Yes (7/2)	Yes, E1 (7/2)
	RBS + LacI

High/ Low Constitutive Promoters + RBS + Repressor Coding Region

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P66	High + RBS + TetR (P56 +P38)
P67	Low + RBS + TetR (P56 + P39)
P68	High + RBS + cI Lambda (P57 + P38)
P69	Low + RBS + cI Lambda (P57 + P39)
	High + RBS + LacI
	Low + RBS + LacI

High/ Low Constitutive Promoters + RBS + Repressor Coding Region + Terminator(s)

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P70	High + RBS + TetR + Term (P66 + )
P71	Low + RBS + TetR + Term (P67 + P39)
P72	High + RBS + cI Lambda + Term (P68 + P38)
P73	Low + RBS + cI Lambda + Term (P69 + P39)
	High + RBS + LacI + Term
	Low + RBS + LacI + Term

RBS + GFP + Term

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P45	GFP only w/ RBS & terminator	N/A	N/A	N/A	N/A	Yes (7/1)	Yes

p-lambda/pTet/pLac Promoter + RBS + GFP + Term

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P15	pTet + RBS + GFP + Term	N/A	N/A	N/A	N/A	Yes	Yes
P20	pLac + RBS + GFP + Term	N/A	N/A	N/A	N/A	Yes	Yes
P74	pLambda + RBS + GFP + Term (P18 + P45)

pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P58	pTet + RBS + GFP + Term in p15a ori (P1 + P15)	Yes (7/1)	Yes (7/2)	Yes (7/2)	Yes (7/2)
P59	pLac + RBS + GFP + Term in p15a ori (P1 + P20)	Yes (7/1)	Yes (7/2)	Yes (7/2)	Yes (7/2)
P75	pLambda + RBS + GFP + Term in p15a ori (P1 + P74)

High/ Low Constitutive Promoters + RBS + Repressor Coding Region + Terminator(s) + pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
	High + RBS + TetR + Term + pTet + RBS + GFP + Term in p15a ori (P70 + P58)
	Low + RBS + TetR + Term + pTet + RBS + GFP + Term in p15a ori (P71 + P58)
	High + RBS + cI Lambda + Term + pLambda + RBS + GFP + Term in p15a ori (P72 + P75)
	Low + RBS + cI Lambda + Term + pLambda + RBS + GFP + Term in p15a ori (P73 + P75)
	High + RBS + LacI + Term + pLac + RBS + GFP + Term in p15a ori (+ P59)
	Low + RBS + LacI + Term + pLac + RBS + GFP + Term in p15a ori (+ P59)

Making a new CDF vector

CDF ori as an insert

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P76	modified P13 + P1 (CDF ori + p15a vector)	P1- 7/2, modified P13- 7/3

CDF ori + Resistance Cassettes as inserts

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P79	P76 + P48 (CDF ori on p15a vector + Cm Resistance)	P48- 7/3
P80	P76 + P49 (CDF ori on p15a vector + Amp Resistance)	P49- 7/3

New Vector w/ CDF ori + Resistance Marker

Plasmid Number	Plasmid Description	Components Digested?	Components Purified?	Ligated?	Transformed?	Miniprepped?	Direct from Registry?
P81	P79 + P5 (CDF ori + Cm resistance on pSB3K3 vector)
P82	P80 + P5 (CDF ori + Amp resistance on pSB3K3 vector)

Transformations from the Registry

Re-Transformation of BioBrick Parts in E1 and E3 (6/27-7/1)

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)

Transformation of Inverters and Repressors (7/1-)

Name	Registry Name	Description	Origin	Size	Marker	Transformed? (7/2)	Miniprepped?	Culture in Glycerol Stock?	Verified via sequencing?
P51	BBa_Q04121	LacI QPI w/ RBS & promoter		1370	Kan	Failed in E1.
P52	BBa_Q04510	cI QPI w/ RBS & promoter		987	Kan	Failed in E1.
P53	BBa_P0440	PoPS --> TetR		840	Amp	Yes, in E1.
P54	BBa_P0412	PoPS --> LacI		1308	Amp	Yes, in E1.
P55	BBa_P0455	RBS.Lambda.cI. LVA.Terminator		896	A/C	Yes, in E1.

Transformation of Terminators and LacI coding regions (7/2)

Name	Registry Name	Description	Origin	Size	Marker	Transformed? (7/2)	Miniprepped?	Culture in Glycerol Stock?	Verified via sequencing?
P41	BBa_B0010	Transcriptional Terminator			Amp	in E1.
P61	BBa_B0011	Terminator				in E1.
P62	BBa_B0012	Terminator				in E1.
P63	BBa_B0014	Double terminator (P62 and P61)				in E1.
P64	BBa_B0015	Double terminator (P60 and P62)				in E1.
P65	BBa_B0025	Double terminator (P62 and P60)				in E1.
P77	BBa_P0312	RBS + LacI + terminators				in E1.
P78	BBa_P0112	RBS + LacI + terminators				in E1.

First Round

Putting Lac/Tet + GFP with P15A, and RBS with TetR and cI lambda coding regions.

In this round:

Vector Name	Vector Description	Insert Name	Insert Description	Projected Resulting Plasmid
P1	P15a ORI	P15	GFP w/ pTet	P58
P1	P15a ORI	P20	GFP with pLac	P59
P40	Vector with RBS	P43	TetR coding region	P56
P40	Vector with RBS	P44	cI lambda coding region	P57

Also cutting P38, P39, and P45 in anticipation of future ligations.

Restriction Enzyme Digestions (7/1)

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 of First Round Digestions (7/1)

File:7-1 digest Biobrick Parts.jpg

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 and Transformation (7/1)

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).

Picking Colonies (7/2)

All four transformations were successful, with many colonies on each plate. Two colonies were picked from each plate.

Minipreps (7/3)

Transformation of P58 and P59, and pET-Duet-1 into Shewanella via Electroporation (7/3)

Second Round

- Adding hi/low constitutive promoters to repressors, OmpR-P promoter to p15a, and p-lambda to (RBS + GFP)

Vector Name	Vector Description	Insert Name	Insert Description	Projected Resulting Plasmid
P1	P15a ORI	P26	Mutated promoter activated by OmpR-P with the reporter GFP	P60
P45	GFP only w/ RBS & terminator	P18	pLambda (cI regulated)	P74
P56	RBS + TetR (P40+P43)	P38	High constitutive promoter	P66
P56	RBS + TetR (P40+P43)	P39	Low constitutive promoter	P67
P57	RBS + cI lambda (P40+P44)	P38	High constitutive promoter	P68
P57	RBS + cI lambda (P40+P44)	P39	Low constitutive promoter	P69
P1	P15a ORI	modified P13	pCDF-Duet 1 (origin PCR)	P76

(Projected Third Round)

Vector Name	Vector Description	Insert Name	Insert Description	Projected Resulting Plasmid
P76	CDF ori in p15a vector	P48	Cm Resistance Cassette	P79 p15a vector w/ CDF ori + Cm resistance
P76	CDF ori in p15a vector	P49	Amp Resistance Cassette	P80 p15a vector with CDF ori + Amp resistance

(Projected Fourth Round)

Vector Name	Vector Description	Insert Name	Insert Description	Projected Resulting Plasmid
P5	Vector w/ NheI sites flanking ori and resistance marker	P79	CDF ori + Cm resistance	P81 (Vector with CDF ori, Cm resistance, and death gene)
P5	Vector w/ NheI sites flanking ori and resistance marker	P80	CDF ori + Amp resistance	P82 (Vector with CDF ori, Amp resistance, and death gene)

Digestions (7/3)

Digestion Reactions:

First Batch

'	P26A (insert)	P48B (07) (insert)	P49A (08) (insert)	pCDF (origin PCR) (insert)
DNA	5 uL	5 uL	5 uL	5 uL
10X Buffer (Volume & #)	2.5 uL of 3	2.5 uL of 3	2.5 uL of 3	2.5 uL of 3
50X BSA	0.5 uL	0.5 uL	0.5 uL	0.5 uL
Restriction Enzyme 1	1 uL XbaI	1 uL XbaI	1 uL XbaI	1 uL XbaI
Restriction Enzyme 2	1 uL PstI	1uL PstI	1 uL PstI	1 uL PstI
Water	15 uL	15 uL	15 uL	15 uL
Total Volume	25 uL	25 uL	25 uL	25 uL

Second Batch (We did some of the digestions in the First Batch incorrectly. Only the correct ones are now listed under First Batch.)

'	P39A (Vector - Prefix)	P38A (Vector - Prefix)	P57B (Insert - Suffix)	P56B (Insert - Suffix)	P18D (Vector - Prefix)	P45B (Insert - Suffix
DNA	5 uL	8 uL	10 uL	10 uL	10 uL	5 uL
10X Buffer (Volume & #)	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	2.5 uL of 3
50X BSA	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL	0.5 uL
Restriction Enzyme 1	1 uL SpeI	1 uL SpeI	1 uL XbaI	1 uL XbaI	1 uL SpeI	1 uL XbaI
Restriction Enzyme 2	1 uL PstI	1 uL PstI	1 uL PstI	1 uL PstI	1 uL PstI	1 uL PstI
Water	15 uL	12 uL	10 uL	10 uL	10 uL	15 uL
Total Volume	25 uL	25 uL	30 uL	25 uL	25 uL	25 uL

All of these parts were run on a gel and purified

Transformation of Parts into S1

Transformation of Lac/Tet + GFP into S1(7/3)

25 mL culture of S1 was grown for electroporation and transformation.

Plasmid Name	uL DNA transformed	Plates grew?	Picked colonies?	Miniprepped?	Glycerol Stocks?
P58B	8uL	many small colonies, no GFP
P59B	8uL	5 medium-sized orangish-pinkish colonies, GFP +
P12 (from Christina)	5 uL
P27	2uL	9 small/medium orangish colonies, also many very small white-ish colonies
P28	2uL	many tiny colonies, no GFP
P29	5uL	many tiny colonies, no Venus fluor
P30	5uL	2 small pinkish colonies, YFP+
P31	8uL
P32	5uL	many (>50) medium orangish-pinkish colonies, no YFP

Team:Harvard/Dailybook/Week2/Chemical and Light