IGEM:Imperial/2010/AmyE Vector

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 : 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

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

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!) Test digests of K54 K70 Midi prep using i) EcoRI, ii) SpeI and iii) EcoRI + SpeI Screen next 20 colonies by colony PCR, use higher temperatures to avoid previous non specific annealing	Miniprep of 4 overnight cultures (dif P and 3' Insert for PyrD) - colonies 4,5,7 & 9 Run Colony PCR results on a Gel - pick promissing candidates for mini prep.
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 Test Digest of Mini Prep K02+dif EcoRI and SpeI	Midiprep of Colony 4 - concentration 110 ng/ul Restriction digest of K02 from registrty for 3A assembly.	Gel purification of insert (35 ul) Gel analysis of vector and insert to work out ratios for ligations	Transformation with overnight ligations PCR amplified the PSB1C3 vector for 3A assembly Gel Purified K02 and PBB1C3 digests	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 Dephosphorylate PSB1C3 using alkaline phosphotase
AFTERNOON	Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow Second Test digest SpeI PME - no positive results. Decided to repeat the step using 3A assembly method to reduce background from vector.	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) Midi prep of sample 8 K54 + K70	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 set up ligation reaction K02+dif using 3A method for Transformation on Monday.