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

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==Transformation of Terminators and LacI coding regions==
==Transformation of Terminators and LacI coding regions (7/2)==
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Name'''
| align="center" style="background:#f0f0f0;"|'''Name'''
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| align="center" style="background:#f0f0f0;"|'''Verified via sequencing?'''
| align="center" style="background:#f0f0f0;"|'''Verified via sequencing?'''
|-
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| P41 ||BBa_B0010||Transcriptional Terminator||||||Amp||||||||
| P41 ||BBa_B0010||Transcriptional Terminator||||||Amp||in E1.||||||
|-
|-
| P61||BBa_B0011||Terminator||||||||||||||
| P61||BBa_B0011||Terminator||in E1.||||||||||||
|-
|-
| P62||BBa_B0012||Terminator||||||||||||||
| P62||BBa_B0012||Terminator||in E1.||||||||||||
|-
|-
| P63||BBa_B0014||Double terminator (P62 and P61)||||||||||||||
| P63||BBa_B0014||Double terminator (P62 and P61)||in E1.||||||||||||
|-
|-
| P64||BBa_B0015||Double terminator (P60 and P62)||||||||||||||
| P64||BBa_B0015||Double terminator (P60 and P62)||in E1.||||||||||||
|-
|-
| P65||BBa_B0025||Double terminator (P62 and P60)||||||||||||||
| P65||BBa_B0025||Double terminator (P62 and P60)||in E1.||||||||||||
|-
|-
| P77||BBa_P0312||RBS + LacI + terminators||||||||||||||
| P77||BBa_P0312||RBS + LacI + terminators||in E1.||||||||||||
|-
|-
| P78||BBa_P0112||RBS + LacI + terminators
| P78||BBa_P0112||RBS + LacI + terminators||in E1.||||||||||||
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Revision as of 19:51, 2 July 2008

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

Scoreboard

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 (7/2-)

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)

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) P76
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

Digestions (7/3)

Digestion Reactions:

' P56 (vector) P57 (vector) P18 (insert) P1 (vector) P26 (insert) P45 (vector) P38 (insert) P39 (insert)
DNA 15 uL 15 uL 15 uL 15 uL 15 uL 15 uL 15 uL 15 uL
10X Buffer (Volume & #) 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 2.5 uL of 2 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
Restriction Enzyme 1 0.5 uL EcoRI 0.5 uL EcoRI 0.5 uL EcoRI 0.5 uL XbaI 0.5 uL XbaI 0.5 uL EcoRI 0.5 uL EcoRI 0.5 uL EcoRI
Restriction Enzyme 2 0.5 uL XbaI 0.5 uL XbaI 0.5 uL SpeI 0.5 uL PstI 0.5 uL PstI 0.5 uL XbaI 0.5 uL SpeI 0.5 uL SpeI
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
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
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