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

From OpenWetWare
Jump to: navigation, search

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

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)

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