Team:Harvard/Dailybook/Week2/Chemical and Light

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Contents

Goals for Week 2

  1. Transform LacI, TetR, cI + their promoters and GFP. Refer http://partsregistry.org/Part:BBa_Q04121 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 http://partsregistry.org/Part:BBa_R0051 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

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?
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)

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 pSB3K3Low-Medium copy vector (w/ death gene)p15a2750Kanin E3YesYes (B- done 6/30. A-done 7/1)
P37 BBa_I722007Constructive expression LacI w/ pTetR promoterAmpFailed (liquid cultures didn\'t grow)
P38 BBa_J23114High constitutive promoter35Amp2007 in E1 (DH5a), 2008 FailedYes.2007 (B- done 6/30. A- done 7/1)
P39 BBa_J23113Low constitutive promoter35Amp2008 (A grew), 2007 (A grew)Yes.2008 (A- done 6/30) 2007 (A-done 6/30)
P40 BBa_B0032Ribosome Binding Site13Amp2008 (A grew), 2007 (B grew)Yes.2007 (B -done 6/30) 2008 (A-done 6/30)
P41 BBa_B0010Transcriptional TerminatorAmpFailed (liquid cultures didn\'t grow)
P42 BBa_C0012LacI coding regionAmpFailed (liquid cultures didn\'t grow)
P43 BBa_C0040TetR coding region660Amp2007 (A grew)Yes2007 (A-done 6/30)
P44 BBa_C0051cI lambda750Amp2007 (A grew)Yes2007 (A-done 6/30)
P45 BBa_E0240GFP only w/ RBS & terminatorAmp2007 (A and B grew)Yes2007 (A and B -done 6/30)
P46 BBa_I51020Base VectorAmpA and B grew.YesA and B -done 6/30
P47 BBa_P1003Kan Resistance Cassette967Kan2007 (A and B grew, but spilled)Yes2007 (A and B -done 7/1)
P48 BBa_P1004Cm Resistance Cassette769Cm2007 (A and B grew, but spilled)Yes2007 (A and B -done 7/1)
P49 BBa_P1002Amp Resistance Cassette943Amp2008 (A grew), 2007 (A and B grew)Yes2008 (A-done 6/30) 2007 (A and B -done 6/30)
P50 BBa_P1005Tet Resistance Cassette1283Tet2007 (A and B grew)Yes2007 (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?
P51BBa_Q04121LacI QPI w/ RBS & promoter1370KanFailed in E1.
P52BBa_Q04510cI QPI w/ RBS & promoter987KanFailed in E1.
P53BBa_P0440PoPS --> TetR840AmpYes, in E1.
P54BBa_P0412PoPS --> LacI1308AmpYes, in E1.
P55BBa_P0455RBS.Lambda.cI. LVA.Terminator896A/CYes, 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_B0010Transcriptional TerminatorAmpin E1.
P61BBa_B0011Terminatorin E1.
P62BBa_B0012Terminatorin E1.
P63BBa_B0014Double terminator (P62 and P61)in E1.
P64BBa_B0015Double terminator (P60 and P62)in E1.
P65BBa_B0025Double terminator (P62 and P60)in E1.
P77BBa_P0312RBS + LacI + terminatorsin E1.
P78BBa_P0112RBS + LacI + terminatorsin 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
P1P15a ORIP15GFP w/ pTetP58
P1P15a ORIP20GFP with pLacP59
P40Vector with RBSP43TetR coding regionP56
P40Vector with RBSP44cI lambda coding regionP57

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)
DNA15 uL15 uL15 uL15 uL15 uL15 uL15 uL15 uL15 uL
10X Buffer (Volume & #)2.5 uL of 32.5 uL of 32.5 uL of 32.5 uL of 22.5 uL of 22.5 uL of 22.5 uL of 32.5 uL of 32.5 uL of 2
50X BSA0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL
Restriction Enzyme 10.5 uL XbaI0.5 uL XbaI0.5 uL XbaI0.5 uL SpeI0.5 uL SpeI0.5 uL SpeI0.5 uL XbaI0.5 uL XbaI0.5 uL SpeI
Restriction Enzyme 20.5 uL PstI0.5 uL PstI0.5 uL PstI0.5 uL PstI0.5 uL PstI0.5 uL PstI0.5 uL PstI0.5 uL PstI0.5 uL PstI
Water6.25 uL6.25 uL6.25 uL6.25 uL6.25 uL6.25 uL6.25 uL6.25 uL6.25 uL
Total Volume25.25 uL25.25 uL25.25 uL25.25 uL25.25 uL25.25 uL25.25 uL25.25 uL25.25 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
1P1
2P15
3P20
41 kB Ladder

Gel 2

Well Sample
1P40
2P43
3P44
4P45
51 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
P1P15a ORIP26Mutated promoter activated by OmpR-P with the reporter GFPP60
P45GFP only w/ RBS & terminatorP18pLambda (cI regulated)P74
P56RBS + TetR (P40+P43)P38High constitutive promoterP66
P56RBS + TetR (P40+P43)P39Low constitutive promoterP67
P57RBS + cI lambda (P40+P44)P38High constitutive promoterP68
P57RBS + cI lambda (P40+P44)P39Low constitutive promoterP69
P1P15a ORImodified P13pCDF-Duet 1 (origin PCR)P76

(Projected Third Round)

Vector Name Vector Description Insert Name Insert Description Projected Resulting Plasmid
P76CDF ori in p15a vectorP48Cm Resistance CassetteP79 p15a vector w/ CDF ori + Cm resistance
P76CDF ori in p15a vectorP49Amp Resistance CassetteP80 p15a vector with CDF ori + Amp resistance

(Projected Fourth Round)

Vector Name Vector Description Insert Name Insert Description Projected Resulting Plasmid
P5Vector w/ NheI sites flanking ori and resistance markerP79CDF ori + Cm resistanceP81 (Vector with CDF ori, Cm resistance, and death gene)
P5Vector w/ NheI sites flanking ori and resistance markerP80CDF ori + Amp resistanceP82 (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)
DNA5 uL5 uL5 uL5 uL
10X Buffer (Volume & #)2.5 uL of 32.5 uL of 32.5 uL of 32.5 uL of 3
50X BSA0.5 uL0.5 uL0.5 uL0.5 uL
Restriction Enzyme 11 uL XbaI1 uL XbaI1 uL XbaI1 uL XbaI
Restriction Enzyme 21 uL PstI1uL PstI1 uL PstI1 uL PstI
Water15 uL15 uL15 uL15 uL
Total Volume25 uL25 uL25 uL25 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
DNA5 uL8 uL10 uL10 uL10 uL5 uL
10X Buffer (Volume & #)2.5 uL of 22.5 uL of 22.5 uL of 32.5 uL of 32.5 uL of 22.5 uL of 3
50X BSA0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL0.5 uL
Restriction Enzyme 11 uL SpeI1 uL SpeI1 uL XbaI1 uL XbaI1 uL SpeI1 uL XbaI
Restriction Enzyme 21 uL PstI1 uL PstI1 uL PstI1 uL PstI1 uL PstI1 uL PstI
Water15 uL12 uL10 uL10 uL10 uL15 uL
Total Volume25 uL25 uL30 uL25 uL25 uL25 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?
P58B8uLmany small colonies, no GFP
P59B8uL5 medium-sized orangish-pinkish colonies, GFP +
P12 (from Christina)5 uL
P272uL9 small/medium orangish colonies, also many very small white-ish colonies
P282uLmany tiny colonies, no GFP
P295uLmany tiny colonies, no Venus fluor
P305uL2 small pinkish colonies, YFP+
P318uL
P325uLmany (>50) medium orangish-pinkish colonies, no YFP
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