Team:Imperial College London/Lab Diaries/Vectors team

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.

• Restriction digest of 5' dif XP with XbaI and PstI

Start assembly of PyrD vector
• Overnight annealing of 5' Ins ( synthesized oligos )

• Gel analysis of resultant products from 5' dif XP digest
• PCR purification of cut 5' dif XP

• Restriction digestion of 5' ins [K143008] (using Eco and Spe)
• PCR amplification of vector backbone pSB1C3

• Gel analysis and extraction of 5' ins
• Gel purification of 5' ins
• Restriction digestion of pSB1C3 (using Eco and Pst)
• PCR purification of pSB1C3 after digestion
• Gel analysis of 5' ins, dif and pSB1C3 to work out ratios for ligation
• Restriction digestion of pveg promoter and RBS [K143053] (using Eco and Spe)
• Restriction digetion of SpecR-T [K143065] (using Xba and Pst)

• Replica plating and colony PCR of transformed colonies (containing 5' dif in pSB1C3)
• Gel extraction and PCR purification of pveg and SpecR-T in preparation for ligations this afternoon
• Gel analysis of pveg , Spec-T and pSB1C3 in order to work out ratios for the ligation

• Transformation of overnight ligations: 5' ins, dif with pSB1C3 and pveg, SpecR-T and pSB1C3

• Replica plating and colony PCR of: 5' ins, dif with pSB1C3 and pveg, SpecR-T and pSB1C3

• PCR purification of PSB1C3 vector

• Ligation of 5' ins and dif with pSB1C3 - a bench ligation (1 hour) and an overnight ligation were set up
• Transformation of E.Coli with the bench ligated products
• Gel analysis and extraction of pveg and SpecR-T

• Overnight ligation of pveg and SpecR-T with pSB1C3

• Gel analysis of colony PCR products from the transformations
• Overnight annealing of diff PES (Synthesized oligo)

• Set up overnight ligations for standard assembly (BBA) and 3A cloning (3A) of dif P
• Restriction digestion of 3' ins in the pSB1AK3 vector ( Using Eco and Xba) for BBA
• PCR purification of 3' ins in AK3 ( Using Eco and Xba) for BBA
• Set up 5 ml culture of pveg, SpecR-T in pSB1C3 from colony 1 of replica plate and kept in shaking incubator at 37 degrees

• Restriction digestion of 3' ins in the AK3 vector ( Using Xba and Pst) for 3A

• Overnight ligation of dif P and 3' ins with pSB1C3
• 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.

• Replica plating of transformed colonies having dif P with 3' ins in AK3 -45 sigle colonies were plated
• The first 15 of the above colonies were used for colony PCR reactions 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

• Miniprep of 4 overnight cultures - diff P with 3' ins in AK3; colonies 4,5,7 & 9

• Gel analysis of PCR purified 3' ins in AK3 and gel purified 3' diff P to work out ratios for the liagation
• Dephosphorylation of digested AK3 vector with 3' ins
• Overnight ligation of dif P with 3' ins in AK3 (insert and vector) and AK3 containing 3' ins vector only
• Set up 100 ml culture of pveg and Spec-T in pSB1C3 by transferring 5 ml culture set up in the morning and kept in shaking incubator at 37 degrees

• Transformation of E.Coli with ligates from yesterday in AmpR;dif P with 3' ins in AK3 (insert and vector) and AK3 containing 3' ins vector only
• Gel extraction of 3' ins (now our insert) described this morning for 3A
• Gel analysis of gel extracted dif P and 3' ins (inserts) and pSB1C3 (vector) to work out ratios for the ligation
• Midiprep of pveg, SpecR-T in pSB1C3 from colony

• Gel analysis of gel extracted dif P and 3' ins (inserts) and pSB1C3 (vector) to work out ratios for the ligation
• pveg, SpecR-T in pSB1C3 midiprep sent for sequencing

• Set up overnight 5 ml cultures containing dif P with 3' ins in AK3 for miniprep 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 containing dif P with 3' ins in AK3 - Two digests : One with Spe & Pst and other with Xba & Spe

• 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

• 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 & Ps
• 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

• Colony PCR of 10 transformed C-Spec colonies
• Set up 5ml culture of N-Spec in KanR

• Miniprep of C-Spec colonies 2, 5 & 9 in SpecR and CmR
• Digest of minipreps; double digest (Eco and Spe) and triple digest (PmeI, Eco and Spe)
• Gel analysis of midiprepped undigested and digested C-Spec

• Miniprep of C-Spec colonies 2, 5 & 9 in SpecR only and CmR only
• Digest of minipreps; double digest (Eco and Spe) and triple digest (PmeI, Eco and Spe)
• Gel analysis of midiprepped undigested and digested C-Spec

• Start midiprep of C-Spec from colony 2 (isopropanol added elute was refrigerated for 4 hrs)
• Measure concentration of midiprepped N-Spec (732 ng/ul)

• Dilution of N-Spec and C-Spec (4x)
• Double digest of N-Spec and C-Spec (Eco and KpnI)
• Gel analysis of diluted undigested and double digested Specs

• Gel analysis of colony PCR
• Set up 5 ml cultures of C-Spec colonies 2,5 & 9 in SpecR & CmR
• Transfer 5 ml culture to 100 ml culture in KanR (incubated overnight)

• Start midiprep of N-Spec (isopropanol added elute refrigerated overnight)
• Set up 5 ml cultures for C-Spec from colonies 2,5 & 9 (in SpecR only and CmR only)

• Continue midiprep of N-Spec (by ethanol preciptation)
• Set up a 500 ml overnight culture (with SpecR) of C-Spec from colony 2 for a midiprep tomorrow

• Continue midiprep of C-Spec (by ethanol precipitation)
• Digest of midiprepped N-Spec and C-Spec (Eco and Spe)
• Gel analysis of undigested and digested (Eco and KpnI) Specs

• Single digest of diluted C-Spec (Eco only)
• Gel analysis of diluted undigested, single digested (Eco only) and double digested (Eco and KpnI)

• Set up digests of N-Spec and C-Spec (Eco and KpnI)- (CD 1)
• Gel analysis and extraction of both N and C Specs

• Gel purification of N and C Specs from CD 2
• Run both Specs from each of the ligations on a gel

• Backbone PCR of pSB1C3 vector using PFU ultra buffer
• PCR purification of pSB1C3

• Backbone PCR of pSB1C3 using Barns buffer
• Replica plating in SpecR of transformed colonies from the insert and vector ligation and setting up 5 ml cultures in SpecR for transformed colonies

• Gel purification of pSB1C3
• Miniprep of 5 ml overnight cultures of colonies 1, 2 and 3

• Set up digests of N-Spec and C-Spec again, however, with a higher dilution of N-Spec - (CD 2)
• Gel analysis and extraction of N and C Specs
• Gel purification of CD 1

• Dephosphorylate vector (C-Spec) from CD 2
• Set up overnight ligations of Vector only (C-Spec) and Vector and Insert (N-Spec and C-Spec) for transformation

• Transformation of E.Coli with the two Spec final ligates - C-Spec (vector only) and N and C Specs (vector and insert)
• Gel analysis of purified pSB1C3

• Gel analysis and extraction of pSB1C3

• Test digest of final spec minipreps with Eco and Kpn1 and Eco and Spe
• Cloning digest of pSB1C3 with Eco and Pst
• Gel analysis of undigested and digested minipreps and digested pSB1C3

• Set up 5 ml culture for miniprep of C-Spec of colnies 1 and 2 from synthesis in AmpR
• Set up sequencing mixes for 3' dif P Ins in AK3, C-Spec minis from cultures 5 & 9 and final spec mini

• Start midiprep of C-Spec
• Miniprep of C-Spec cultures from yesterday

• Cloning digests of C-Spec with Kpn1 and Pst and N-Spec with Eco and Kpn1
• Gel purification of the inserts

• Miniprep cultures of C-Spec have not grown well enough therefore they will be left overnight
• Set up 100 ml culture for midiprepping tomorrow

• Test digest C-Spec mini with ECo and KpnI
• Run test digest on gel, both minis from colonies 1 & 2 look good, therefore proceed to midiprep
• Continue with midiprep but no pellet after 45 minute spin
• Set up 100 ml cultures of colonies 1 & 2 for midiprep tomorrow

• Continue Midiprep of C-Spec, pellets obtained
• Transformation of E.Coli with the two Spec final ligates - C3 (vector only) and N and C Specs in C3 (vector and insert)

• Ligation gel with inserts; N-Spec (E & Kpn1) and C-Spec (Kpn1 & P) and Vector; pSB1C3 (E & P)
• Dephosphorylation of Vector and overnight ligation of vector alone and vector with inserts

• Transformation of E.Coli with the two Spec final ligates - C3 (vector only) and N and C Specs in C3 (vector and insert)

• Transformations failed, re-try next week!

• Set up 5 ml culture for miniprep of C-Spec of colnies 1 and 2 from synthesis in AmpR
• Set up sequencing mixes for 3' dif P Ins in AK3, C-Spec minis from cultures 5 & 9 and final spec mini

• Start midiprep of C-Spec
• Miniprep of C-Spec cultures from yesterday

• Cloning digests of C-Spec with Kpn1 and Pst and N-Spec with Eco and Kpn1
• Gel purification of the inserts

• Miniprep cultures of C-Spec have not grown well enough therefore they will be left overnight
• Set up 100 ml culture for midiprepping tomorrow

• Test digest C-Spec mini with ECo and KpnI
• Run test digest on gel, both minis from colonies 1 & 2 look good, therefore proceed to midiprep
• Continue with midiprep but no pellet after 45 minute spin
• Set up 100 ml cultures of colonies 1 & 2 for midiprep tomorrow

• Continue Midiprep of C-Spec, pellets obtained
• Transformation of E.Coli with the two Spec final ligates - C3 (vector only) and N and C Specs in C3 (vector and insert)

• Ligation gel with inserts; N-Spec (E & Kpn1) and C-Spec (Kpn1 & P) and Vector; pSB1C3 (E & P)
• Dephosphorylation of Vector and overnight ligation of vector alone and vector with inserts

• Transformation of E.Coli with the two Spec final ligates - C3 (vector only) and N and C Specs in C3 (vector and insert)

• Transformations failed, re-try next week!

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.

K14070 and K14064 fragments

1. DNA was taken out of the reigstry
2. Cut with restriction enzymes
3. Run on a gel to confirm correct cutting and estimate relative ratios of DNA for ligation
4. Ligated overnight
5. Transformed into E.Coli to Amplify the DNA
6. Colony PCR has been used to confirm the correct insert size.

Next Steps:

1. Extract the DNA with a miniprep
2. Proceed to the next step - Reverse PCR

K14002 and oligos

1. Ligated two single stranded oligos together to produce Dif and insertion site
2. Standard biobrick assembly of oligos to K14002
3. Ligation and transformation into E.coli competent cells (strain)
4. Screen plated colonies for correct insertion

Next Steps:

1. Purify the correct insert out of E.coli
2. Next step assembly - LacI test vector and Final assembly vector

LacI Testing vector Currently waiting for Part B step 2 Midi-prep results to start this step.