IGEM:Imperial/2010/Vectors team

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<p id="popshow">Follow our progress: Click me!</p>
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  <li><a href="#Objectives" class="popt">Objectives</a></li>
  <li><a href="#Assembly_of_Vectors" class="popt">Assembly of Vectors</a></li>
  <li><a href="#Schedule" class="popt">Schedule</a></li>
  <li><a href="#Lab_Notes" class="popt">Lab Notes</a></li>

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Objectives

We are assembling the AmyE and PyrD vectors in order to transform B. Subtilis with our parts. Once completed, these vectors will be reusable and can then be used to introduce any relevant piece of DNA directly into B.subtilis genome. The vectors will be used both for the final assembly and for testing constructs.


  • Assemble the PyrD vector
  • Assemble the AmyE vector

Assembly of Vectors

A
Overveiw of PyrD assembly



A
Overveiw of AmyE assembly


Starting from the top, we are assembling the first two fragments (K14070 and K14064) and K147002 with our oligos to add in a Dif site. Two dif sites on either side of resistance cassettes can be used to later excise antibiotic resistance from our final constructs.

A
A : K143070 and K11064 assembly step
  • K14070 and K14064 fragments
- DNA was taken out of the reigstry
- Cut with restriction enzymes
- Run on a gel to confirm correct cutting
  and estimate relative ratios of DNA for ligation
- Ligated overnight
- Transformed into E.Coli to Amplify the DNA
- Colony PCR has been used to confirm the correct insert size.
  • Next Steps:
- Extract the DNA with a miniprep
- Proceed to the next step - Reverse PCR


A
B : K14002 and oligo assembly






  • K14002 and oligos
- Ligated two single stranded oligos together to produce Dif and insertion site
- Standard biobrick assembly of oligos to K14002
- Ligation and transformation into E.coli competent cells (strain)
- Screen plated colonies for correct insertion
  • Next Steps
- Purify the correct insert out of E.coli
- Next step assembly - LacI test vector and Final assembly vector









A
B2 : LacI Testing Vector
  • LacI Testing vector
- Currently waiting for Part B step 2 Midi-prep results to start this step

Schedule & Lab notes

PyrD Vector

Week 6

Week 6 Monday Tuesday Wednesday Thursday Friday
MORNING
  • Restriction digestion of 5' Dif XP ( Using XbaI and PstI)

AFTERNOON

Start assembly of PyrD vector

  • Overnight annealing of the synthesized oligos (5' Ins)
  • Gel analysis of resultant products from 5' Dif XP digestion
  • PCR purification of 5' Dif XP
  • Further gel analysis after purifcation
Thursday, August 12
  • Annealing the forward and reverse strands of the dif XP oligo

The forward and reverse strands of the 5' dif site with XbaI and PstI restriction sites on either side have been synthesized separately. The synthesized fragments arrive in solid powder form. These were immediately diluted in ddH2O to obtain a stock concentration of 1 ng/ul. They were then allowed to anneal together by heating them to 95 degs for denaturation and allowing them to cool down and anneal overnight.

  • Restriction digest of diff XP

After annealing the two strands, the oligo was cut with XbaI and PstI to obtain overhangs that would later ligate with the compatible overhangs of a digested vector.

  • Gel Analysis of diff XP
Friday, August 13

Week 7

Week 6 Monday Tuesday Wednesday Thursday Friday
MORNING Starting assembly of AmyE vector
  • Restriction digestion of BB k14070 for the AmyE vector (using Eco and Spe)
  • Restriction digestion of BB k14064 for the AmyE vector (using Eco and Xba)
  • Restriction digestion of 5' integration site (k08) for the PyrD vector (using Eco and Spe)
  • PCR amplification of vector backbone PSB1C3 for the PyrD vector
  • Gel analysis and extraction of 5' int site for PyrD vector (repetition of step due to absence of DNA during gel analysis yesterday)
  • Gel purification of k70
  • Gel purification of k08
  • Re-analysis of k70, k64 (for AmyE) on gel to work out ratios for ligation set up
  • Re-analysis of k08, Pme oligos and pSB1C3 (for PyrD) on gel to work out ratios for ligation set up
  • Restriction digestion of pveg promoter (k53) (using Eco and Spe) and spec cassette (k65) (using Xba and Pst) for PyrD vector
  • Restriction digestion of pSB1C3 for PyrD (using Eco and Pst)
  • Check for transformed colonies (colonies that have taken up the vector with the 5'diff) and prepare for colony PCR
  • Gel purification of k53 and k65 for AmyE in preparation for ligations this afternoon
  • Gel extraction and re-analysis on gel of k53, k65 and psB1C3 for Spec casette in preparation for ligations this afternoon
  • Transformation of overnight ligations of:

k64 and k70, k70 only, Spec and 5' PyrD diff

  • Replica plating and colony PCR of:

Spec and 5' PyrD diff

  • Plate wash of:

k64 and k70. k70 only was discarded since this was purely for a baground check

AFTERNOON
  • PCR purification of PSB1C3 vector
  • PCR purification of BB k14064 digestion products
  • Gel analysis of digestion products of BB k14070
  • Gel extraction of digestion products of BB k14070
  • Restriction digestion of k64 and subsequent PCR purification (repetition of step due to absence of DNA during gel analysis)
  • Gel analysis of k70, k64 (for AmyE) to work out ligation ratios
  • Gel analysis and extraction of k53 and k65
  • Bench (1 hour) and overnight ligation of 5'diff with the pSB1C3 vector
  • Transformation of E.Coli with the bench ligated vector
  • Dephosphorylation of k64 and set up of overnight ligations for k64 and k70 (vector and insert) and k64 (vector)only (for negative control; check of background)
  • Set up overnight ligations of SpecR
  • Gel analysis of colony PCR products of Spec and 5' PyrD diff
  • Annealing of diff P oligos (used for both PyrD and AmyE vectors)

Week 6

Week 6 Monday Tuesday Wednesday Thursday Friday
MORNING
  • Restriction digestion of 5' integration site for the PyrD vector ( Using Spe and Pst)

AFTERNOON
  • Starting assembly of PyrD vector
  • Annealing the synthesized oligos ( Pme and diff sites)
  • Gel analysis of resultant products from digestion mixture
  • PCR purification of digestion mixture
  • Further gel analysis

Week 7

Week 6 Monday Tuesday Wednesday Thursday Friday
MORNING Starting assembly of AmyE vector
  • Restriction digestion of BB k14070 for the AmyE vector (using Eco and Spe)
  • Restriction digestion of BB k14064 for the AmyE vector (using Eco and Xba)
  • Restriction digestion of 5' integration site (k08) for the PyrD vector (using Eco and Spe)
  • PCR amplification of vector backbone PSB1C3 for the PyrD vector
  • Gel analysis and extraction of 5' int site for PyrD vector (repetition of step due to absence of DNA during gel analysis yesterday)
  • Gel purification of k70
  • Gel purification of k08
  • Re-analysis of k70, k64 (for AmyE) on gel to work out ratios for ligation set up
  • Re-analysis of k08, Pme oligos and pSB1C3 (for PyrD) on gel to work out ratios for ligation set up
  • Restriction digestion of pveg promoter (k53) (using Eco and Spe) and spec cassette (k65) (using Xba and Pst) for PyrD vector
  • Restriction digestion of pSB1C3 for PyrD (using Eco and Pst)
  • Check for transformed colonies (colonies that have taken up the vector with the 5'diff) and prepare for colony PCR
  • Gel purification of k53 and k65 for AmyE in preparation for ligations this afternoon
  • Gel extraction and re-analysis on gel of k53, k65 and psB1C3 for Spec casette in preparation for ligations this afternoon
  • Transformation of overnight ligations of:

k64 and k70, k70 only, Spec and 5' PyrD diff

  • Replica plating and colony PCR of:

Spec and 5' PyrD diff

  • Plate wash of:

k64 and k70. k70 only was discarded since this was purely for a baground check

AFTERNOON
  • PCR purification of PSB1C3 vector
  • PCR purification of BB k14064 digestion products
  • Gel analysis of digestion products of BB k14070
  • Gel extraction of digestion products of BB k14070
  • Restriction digestion of k64 and subsequent PCR purification (repetition of step due to absence of DNA during gel analysis)
  • Gel analysis of k70, k64 (for AmyE) to work out ligation ratios
  • Gel analysis and extraction of k53 and k65
  • Bench (1 hour) and overnight ligation of 5'diff with the pSB1C3 vector
  • Transformation of E.Coli with the bench ligated vector
  • Dephosphorylation of k64 and set up of overnight ligations for k64 and k70 (vector and insert) and k64 (vector)only (for negative control; check of background)
  • Set up overnight ligations of SpecR
  • Gel analysis of colony PCR products of Spec and 5' PyrD diff
  • Annealing of diff P oligos (used for both PyrD and AmyE vectors)

Week 8

Week 8 Monday Tuesday Wednesday Thursday Friday Saturday
MORNING

Set up overnight ligations for standard assembly (BBA) and 3A cloning (3A) of dif P

  • Restriction digestion of 3' integration site (K02) in the A2 vector ( Using Eco and Xba) for BBA

  • Restriction digestion of 3' integration site (K09) in the AK3 vector ( Using Eco and Xba) for BBA

  • PCR purification of 3' integration site (K02) in the A2 vector ( Using Eco and Xba) for BBA

  • PCR purification of 3' integration site (K09) in the AK3 vector ( Using Eco and Xba) for BBA

  • Set up 5 ml culture of spec from colony 1 of replica plate in shaking incubator @ 37 degrees
  • Restriction digestion of 3' integration site (K02) in the A2 vector ( Using Xba and Pst) for 3A

  • Restriction digestion of 3' integration site (K09) in the AK3 vector ( Using Xba and Pst) for 3A
  • Transformations using the overnight ligations showed a lot of background. Therefore we set up ligations for K02 and K09 using 3A cloning. The results will show if this method is preferable due to less background.
  • Midiprep of CmR vector and test digest using Eco and Spe
  • Backbone PCR of PSB1C3 vector (1st attempt) for common use
  • Replica plating of transformed colonies for k09 from the plate with the insert (diff P) - 45 sigle colonies were plated
  • The first 15 of the above colonies were colony PCRed using dif PES Fwd and pSB Rev
  • Gel analysis of colony PCR from yesterday (first 15 replica plated colonies
  • Set up further colony PCR reactions for the same 15 colonies using pSB Fwd and pSB Rev primers
  • Gel analysis of pSB1C3 - after backbone PCR and after digestion (looked contaminated!)
  • Miniprep of 4 overnight cultures (dif P and 3' Insert for PyrD) - colonies 4,5,7 & 9
AFTERNOON
  • Gel analysis of PCR purified K02 and K09 with the insert (diff P) in between to work out ratios for the liagation
  • Dephosphorylation of digested A2 vector with 3' integration site (K02)

  • Dephosphorylation of digested AK3 vector with 3' integration site (K09)

  • Set up overnight ligation of A2 vector with 3' integration site (K02) with diff P (insert) and A2 vector only

  • Set up overnight ligation of AK3 vector with 3' integration site (K09) with diff P (insert) and AK3 vector only
  • Colony PCR and gel analysis of plated culture (from plate wash on Friday) with K64 and K70
  • Overnight 100 ml culture of spec @ 14 degrees
  • Electroporation of the 4 overnight ligations described on Thursday afternoon
  • Gel extraction of the digestion products ( 3' integration sites - now our inserts) described this morning for 3A
  • Gel analysis of gel extracted K02 nad K09 (inserts) with diff P (also an insert) and pSB1C3 (vector) in between
  • Set up overnight 100 ml culture of CmR vector (K64 and K70) for midiprep tomorrow
  • Set up overnight culture plates (AmpR) for the 4 electroporated cultures (colonies that survive will contain transformed cells
  • PCR purification of PSB1C3 PCR amplified vector
  • Check concentration of midi prepped CmR
  • Run a gel to visualise the results - Gel contained pSB1C3 (PCR purified) , CmR (Midiprepped) and CmR digested (Midiprepped)
  • Backbone PCR of pSB1C3 (2nd attempt), PCR purified and then digested with Eco and Pst
  • Midipreps sent for sequencing ( Spec and CmR)

Backbone PCR of pSB1C3 using PFU (3rd attempt)

  • Set up overnight 5 ml cultures for miniprepping tomorrow - 4 cultures were set up by looking at the gel this morning; 2 positive looking (4 & 7), 1 negative (5) and one containing nothing (9)
  • Diagnostic digests of minipreps - Two digests : One with Spe & Pst and other with Xba & Spe

Week 9

Week 8 Monday Tuesday Wednesday Thursday Friday
MORNING
  • Gel analysis of Mini preps and diagnostic digests from Saturday - Colony 4 looked best
  • Midiprep of Colony 4 - concentration 110 ng/ul
  • Gel purification of insert (35 ul)
  • Gel analysis of vector and insert to work out ratios for ligations
  • Transformation with overnight ligations
  • The transformations were highly successful!! The Vector only plate showed no colonies and the Insert & Vector plate showed many individual colonies
  • 10 individual colonies were replica plated and used for colony PCR
AFTERNOON
  • Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow
  • Digests set up for Vector (SpecR) with Spe & Pst and Insert (dif P & K09)with Xba & Pst
  • PCR purification of vetor (35 ul)
  • Gel extraction of insert after gel analysis (35 ul)
  • Dephosphorylation of vector
  • Set up two overnight ligations; Vector & Insert and Vector only
  • Both ligations (Insert & Vector and Vector only) were plated in CmR and incubated overnight
  • Gel analysis of colony PCR products

Lab Notes

Thursday, 12 August

Friday, 13 August

Monday, 16 August

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