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

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(Transformation of Lac/Tet + GFP into S1)
(Ligations 07/09/08)
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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).
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).
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==07/10/08==
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*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.

Revision as of 11:14, 10 July 2008

Contents

Goals for Week 3

Summary Powerpoint by Amy™

Image: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 have and don't have

RBS + Repressor Coding Region

Plasmid Number Plasmid Description Components Digested? Components Purified? Ligated? Transformed? Miniprepped? Direct from Registry?
P56RBS + TetR (P40+P43)Yes (7/1)Yes (7/2)Yes (7/2)Yes, in E1 (7/2)
P57RBS + 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?
P66High + RBS + TetR (P56 +P38)
P67Low + RBS + TetR (P56 + P39)
P68High + RBS + cI Lambda (P57 + P38)
P69Low + 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?
P70High + RBS + TetR + Term (P66 + )
P71Low + RBS + TetR + Term (P67 + P39)
P72High + RBS + cI Lambda + Term (P68 + P38)
P73Low + 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?
P45GFP only w/ RBS & terminatorN/AN/AN/AN/AYes (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?
P15pTet + RBS + GFP + TermN/AN/AN/AN/AYes Yes
P20pLac + RBS + GFP + TermN/AN/AN/AN/AYes Yes
P74pLambda + 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?
P58pTet + RBS + GFP + Term in p15a ori (P1 + P15)Yes (7/1)Yes (7/2)Yes (7/2)Yes (7/2)
P59pLac + RBS + GFP + Term in p15a ori (P1 + P20)Yes (7/1)Yes (7/2)Yes (7/2)Yes (7/2)
P75pLambda + 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?
P76modified 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?
P79P76 + P48 (CDF ori on p15a vector + Cm Resistance)P48- 7/3
P80P76 + 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?
P81P79 + P5 (CDF ori + Cm resistance on pSB3K3 vector)
P82P80 + 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?
P58B8uLmany small colonies, no GFPYesno growth
P59B8uL5 medium-sized orangish-pinkish colonies, GFP +Yesyes
P12 (from Christina)5 uLlots of small, but pinkish coloniesYesyes
P272uL9 small/medium orangish colonies, also many very small white-ish coloniesYesyes
P282uLmany tiny colonies, no GFPYesno growth
P295uLmany tiny colonies, no Venus fluorYesno growth
P305uL2 small pinkish colonies, YFP+Yesyes
P318uLlots of very small colonies, no VenusYes
P325uLmany (>50) medium orangish-pinkish colonies, no YFPYesyes

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
DNA10 μL20 μL3 μL5 μL5 μL3 μL10 μL10 μL
10X NEB Bufferbuffer 3, 2.5 μLbuffer 2, 2.5 μL buffer 3, 2.5 μLbuffer 2, 2.5 μLbuffer 2, 2.5 μLbuffer 3, 2.5 μLbuffer 3, 2.5 μLbuffer 3, 2.5 μL
25X BSA1 μL1 μL1 μL1 μL1 μL1 μL1 μL1 μL
REs (1 μL each)XPSPXPSPSPXPXPXP
Water9.5 μL0 μL16.5 μL14.5 μL14.5 μL16.5 μL9.5 μL9.5 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 84.441.92.13
p30 (S1) 7/9/2008 4:25 PM 40.372.232.19
p84a (E1) 7/9/2008 4:26 PM 291.421.962.23
p84b (E1) 7/9/2008 4:28 PM 349.611.912.03
p84c (E1) 7/9/2008 4:29 PM 237.091.921.98
p85a (E1) 7/9/2008 4:30 PM 327.451.942.12
p85b (E1) 7/9/2008 4:32 PM 331.991.952.1
p18 (E) 7/9/2008 4:33 PM 135.091.971.81
p45 (E) 7/9/2008 4:34 PM 296.221.941.99
p49 (E) 7/9/2008 4:35 PM 614.331.912.16

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.

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)

Image:7-9_gel_1_MXHTA.jpg 3% low melt agarose, visualized using EtBr/UV
Lane Contents
1100 bp ladder
2P38A PCR product with ES: 35 (NO BAND)
3P38B PCR product with XP: 35 (NO BAND)
4P39A PCR product with ES: 35 (NO BAND)
5P39B PCR product with XP: 35 (NO BAND)
6P63 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)
Image:7-9_gel_3_MXHTA.jpg 1.5% low-melt agarose gel visualized using EtBr/UV
Lane Contents
1P11 with ES: 4333 (NO BAND)
2P40 with SP: 2092 (extracted)
3P23 with EX: 2157 (extracted)
blank
4P24 with EX: 2187 (extracted)
blank
5P56 with EX: 2752 (extracted)
6P45A with EX: 2955 (NO BAND)
7P58 with EX: 3016 (extracted)
8P45A with EX: 2955 (NO BAND)
9P59 with EX: 3201 (extracted)
121 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).

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.
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