IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week3/Chemical and Light

Summary Powerpoint by Amy™

File:7-7monmtg slides (compatible).ppt

The Cloning Strategy

Goals:

• Lac Plasmid: Hi/low promoter + RBS + LacI + terminator + pLac + RBS + GFP + terminator
• Tet Plasmid: Hi/low promoter + RBS + TetR + terminator + pTet + RBS + GFP + terminator
• cI Lambda Plasmid: Hi/low promoter + RBS + cI + terminator + pLambda + RBS + GFP + terminator
• Light Plasmid: P11 + ompC/ompF + RBS + GFP + terminator AND P11 + ompC/ompF + RBS
• CDF vector w/ different resistances: New Vector w/ CDF ori + Resistance Marker (and Death Gene)

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

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)

Transformation of Parts/ Plasmids 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	Yes	no growth
P59B	8uL	5 medium-sized orangish-pinkish colonies, GFP +	Yes	yes
P12 (from Christina)	5 uL	lots of small, but pinkish colonies	Yes	yes
P27	2uL	9 small/medium orangish colonies, also many very small white-ish colonies	Yes	yes
P28	2uL	many tiny colonies, no GFP	Yes	no growth
P29	5uL	many tiny colonies, no Venus fluor	Yes	no growth
P30	5uL	2 small pinkish colonies, YFP+	Yes	yes
P31	8uL	lots of very small colonies, no Venus	Yes
P32	5uL	many (>50) medium orangish-pinkish colonies, no YFP	Yes	yes

Kemikale 'n' Lyte

RE digests 07/07/08

Repeated digests of P1, P18, P26, P38, P39, P45, P56, P57

	P1B	P18	P26A	P38B	P39A	P45B	P56B	P57B
DNA	10 μL	20 μL	3 μL	5 μL	5 μL	3 μL	10 μL	10 μL
10X NEB Buffer	buffer 3, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 2, 2.5 μL	buffer 2, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL	buffer 3, 2.5 μL
25X BSA	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL	1 μL
REs (1 μL each)	XP	SP	XP	SP	SP	XP	XP	XP
Water	9.5 μL	0 μL	16.5 μL	14.5 μL	14.5 μL	16.5 μL	9.5 μL	9.5 μL

07/08: Digested pCDF with XP:

• 12.5 μL water
• 1 μL 25X BSA
• 2.5 μL 10X NEB Buffer 3
• 7 μL DNA
• 1 μL of each RE (XbaI and PstI)

07/08: All of these digested plasmids were run on a 1% low melt gel.

MXH's gel extraction protocol:

• Cut out the bands
• Freeze at –20C for at least 20'
• Spin 13000 RPM 10’
• Use supernatant for ligation
• Then do gel extraction on rest of gel (QIAGEN protocol) and combine with above

Here is a ligation protocol (used by TA and MXH):

• 2 μL 5X Dilution Buffer
• 2 μL vector
• 6 μL insert
• 10 μL 2X Rapid Ligation Buffer
• 1 μL ligase
• vortex briefly
• incubate at RT for 10'
• use 5 μL to transform 50 μL cells

Transformations/Minipreps of Parts from Registries 07/07/08

07/07: We transformed P42, P11, P51, P52, P77, P78, P17, Q01121, BBa_J06911, BBa_J06912 from both the 2007 and 2008 registries (P11 was available only from the 2008 registry). We also made a positive transformation control using pUC19 DNA that came with the TOP10 cells. We made three negative plate controls using mock transformed bacteria on Amp, Kan, and Cm plates. We used E1.

FAILED TRANSFORMATIONS

2007 -- P51 (Kan), Q01121 (Kan), P17 (Kan), P52 (Kan), P78 (Kan), P77 (Kan)

2008 -- P52 (Kan), P51 (Kan), P78 (Kan), P77 (Kan), Q01121 (Kan), P17 (Kan), P11 (Cm)

The Cm and Kan mock transformations (negative controls) also failed to grow.

Alarmingly, the E1 mock transformation (no DNA, neg control) grew on the LB Carb plates. All of the Carb plates had some colonies, so we only picked from those with significantly higher numbers than the neg control. 3 colonies each from BBa_J06911 & BBa_J06912 2007 (temp sensitive LacI system) were picked and grown.

Successful Transformations

2007: P84, P85 (BBa_J06911, BBa_J06912) -- miniprepped/glycerol stocks made

The Amp negative control colonies did not grow in liquid LB Amp. The Amp positive control did grow in liquid LB Amp (pUC19).

Nanodrop of Minipreps

Plasmid	Date	Time	ng/ul	260/280	260/230
p59 (S1)	7/9/2008	4:23 PM	84.44	1.9	2.13
p30 (S1)	7/9/2008	4:25 PM	40.37	2.23	2.19
p84a (E1)	7/9/2008	4:26 PM	291.42	1.96	2.23
p84b (E1)	7/9/2008	4:28 PM	349.61	1.91	2.03
p84c (E1)	7/9/2008	4:29 PM	237.09	1.92	1.98
p85a (E1)	7/9/2008	4:30 PM	327.45	1.94	2.12
p85b (E1)	7/9/2008	4:32 PM	331.99	1.95	2.1
p18 (E)	7/9/2008	4:33 PM	135.09	1.97	1.81
p45 (E)	7/9/2008	4:34 PM	296.22	1.94	1.99
p49 (E)	7/9/2008	4:35 PM	614.33	1.91	2.16

Part PCRs

07/07: More CDF was PCRed (w/ BioBrick adapting primers): 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 @ 58.7°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid

07/08: P11 and P17 from 2008 (left from punch), and P26, P38, P39, P42, P51, P52, P77, P78, Q01121 from 2007 plate were PCRed: 45μL PCR supermix, 1μL DNA, 1μL BBpfx primer (20μM), 1μL BBsfx (20μM), 2μL water. Conditions: 5min @ 94°C → 35x[45s @ 94°C → 45s @ 59°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid We're PCRing these b/c transformation for amplification has not been going smoothly.

07/10: P11: 5min @ 94°C → 40x[45s @ 94°C → 45s @ 56°C → 4m47s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid

Ligations

mtrA and p45

7/7/08 Ligated mtrA and p45 using standard protocol from ligation kit. In step 2 5uL of Vector DNA was added to make a 20uL dephosphorylation mix. In step 6, 4uL of the dephosphorylated Vector DNA and 4uL insert DNA were added.

Mutant Strand Syntheis Reaction

7/8/08 Used Stratagene QuikChange Site-Directed Mutatgenesis Kit (protocol) to create a point mutation in the pstI site in mtrA. Used 5ug of primer.

Gradient PCR

7/8/08 Used gradient PCR to PCR mtrB. Cycled 30-40°C x 10 and then 52-62°C x 30.

RE digests 07/08/08

We attempted to digest the following plasmids (per the Master Plan sent out to the iGEM google group):

• A (BBa_Q01121) with ES
• P11 with ES
• P17 with ES
• P23 with EX
• P24 with EX
• P26 with XP
• P38A with ES
• P38B with XP
• P39A with ES
• P39B with XP
• P42 with XP
• P56 with EX
• P58 with EX
• P59 with EX
• P63 with ES
• P77 with ES
• P78 with ES

These digests were run on gels and the bands of the correct size (when present) were extracted.

Gels (expected sizes of parts indicated)

	3% low melt agarose, visualized using EtBr/UV
	Lane	Contents
	1	100 bp ladder
	2	P38A PCR product with ES: 35 (NO BAND)
	3	P38B PCR product with XP: 35 (NO BAND)
	4	P39A PCR product with ES: 35 (NO BAND)
	5	P39B PCR product with XP: 35 (NO BAND)
	6	P63 with ES: 95 (extracted)

1.5% gel (all of PCR products)

• 100 bp ladder
• A (BBa_Q01121) with ES: 1372 (NO BAND)
• P17 with ES: 902 (NO BAND)
• P26 with XP: 961 (NO BAND)
• P42 with XP: 1128 (NO BAND)
• P77 with ES: 1307 (NO BAND)
• P78 with ES: 1310 (NO BAND)
• CDF with XP: ~900 (NO BAND)

	1.5% low-melt agarose gel visualized using EtBr/UV
	Lane	Contents
	1	P11 with ES: 4333 (NO BAND)
	2	P40 with SP: 2092 (extracted)
	3	P23 with EX: 2157 (extracted)
	blank
	4	P24 with EX: 2187 (extracted)
	blank
	5	P56 with EX: 2752 (extracted)
	6	P45A with EX: 2955 (NO BAND)
	7	P58 with EX: 3016 (extracted)
	8	P45A with EX: 2955 (NO BAND)
	9	P59 with EX: 3201 (extracted)
	12	1 KB ladder

Ligations 07/09/08

We ligated P59 (vector) and P63 (insert); P58 (vector) and P63 (insert); P18 (vector) and P45 (insert).

• 2 μL 5X Dilution Buffer
• 2 μL vector
• 6 μL insert
• 10 μL 2X Rapid Ligation Buffer
• 1 μL ligase
• vortex briefly
• incubate at RT for 10'
• use 5 μL to transform 50 μL competent cells

We transformed the ligated plasmids (P59+63 and P58+63 on Kan, P18+45 on Amp). We also an Amp positive control of pUC19 and two mock transformations (negative controls).

Only the ligation of P18 to P45 (P74) was successful (the plate had colonies that were fluorescent). We made a glycerol stock and a LB amp culture.

07/10/08

• pPL-PCB from UT Austin successfully transformed into Shewanella (p15a ori)
• pCph1-envZ-pprotet did not transform into Shewie (ColE1 ori)
• The ligated p45-mtrA plasmid did not successfully mutate and/or transform into the ultracompetetent cells. This implies that we should focus our efforts on mtrB as it does not require a mutation.

RE digest 07/10/08

Digested P58 and 59 with EX, P45 with XP, P53 and P54 with ES, and P38 and P39 with SP. We plan to ligate

• P53/54 with P58/59. This will give us RBS + LacI/TetR + terminator + pLac/pTet + RBS + GFP + terminator.
• P38/39 with P45. This will give us Hi/low constitutive promoter + RBS + GFP + terminator. This is to test whether we actually have P38 and P39.