Team:Imperial College London/Lab Diaries/Vectors team

From 2010.igem.org

(Difference between revisions)

Revision as of 02:06, 25 October 2010

Department of Bioengineering

Division of Molecular Biosciences

Lab Diaries	Overview \| Surface Protein Team \| XylE Team \| Vectors Team \| Modelling Team
Here are the technical diaries for our project. We've split them up into three lab teams and the modelling team. We think it's really important that absolutely anyone can find out what we've been doing. For a really detailed look at what we did, and when, you've come to the right place!

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.

PyrD Vector

Week 6

Day	Monday	Tuesday	Wednesday	Thursday	Friday	Saturday
Morning					Restriction digest of 5' dif XP with XbaI and PstI
Afternoon					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

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 first heating them to 95 degs for denaturation and allowing them to cool down and anneal overnight.

Friday, August 13

Restriction digest of dif 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 cut vector.

PCR purification of the cut dif XP

The cut oligo was PCR purified in order to get rid of any contaminants. PCR purification gets rid of short pieces of DNA which are less than about 40 base pairs.

Gel Analysis of dif XP

Week 7

Day	Monday	Tuesday	Wednesday	Thursday	Friday
Morning	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
Afternoon	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)

Monday, August 16

Restriction digest of 5' ins [K143008]

K143008 is the 5' integration site for the PyrD vector. This will be used as a front insert together with the dif XP for the pSB1C3 vector.

PCR amplification of pSB1C3

The pSB1C3 vector backbone from the registry was amplified with the use of SB3 and SB2a primers. Submission of parts to the registry requires them to be in a pSB1C3 vector therefore any parts to be submitted will be inserted into this vector.

PCR purification of pSB1C3

The PCR amplified vector was purified in order to get rid of any contaminants. For example, short pieces of DNA like the primers.

Tuesday, August 17

Gel extraction and purification of 5' ins ES

The digested 5' ins was first analyzed on the gel to verify it's size and then extracted for purification. The 5' ins was gel purified in order to extract only the relevant piece of DNA.

Restriction digest of pSB1C3

The pSB1C3 vector was digested so that it would contain compatible overhangs for ligation with inserts.

PCR purification of pSB1C3 EP

The digested pSB1C3 was re-purified in order to get rid of any contaminant DNA that arose during the digestion.

Restriction digests of pveg and Spec-T

pveg (promoter and RBS) and Spec-T (Spectinomycin with a terminator) were digested in preparation for 3A assembly.

Ligation of 5' ins (ES) and dif (XP) with pSB1C3 (EP)

The digested 5' ins and dif (the front inserts) were ligated overnight with pSB1C3 (the vector). A bench ligation and an overnight ligation were set up.

Transformation of E.Coli with bench ligate

E.Coli was transformed via the chemical method using the bench ligate.

Gel extraction and purification of pveg and Spec-T

Since these are both inserts they were gel extracted and purified. PCR purification is not carried out for inserts since they are small and would therefore be lost during the process.

Wednesday, August 18

Replica plating and colony PCR of 5' ins and dif in pSB1C3 (bench ligation)

Ligation of pveg (ES) and SpecR-T (XP) with pSB1C3 (EP)

pveg and SpecR-T (the front inserts) were ligated overnight with pSB1C3 (the vector).

Thursday, August 19

Transformation of E.Coli with the overnights ligates

5' ins and dif in pSB1C3
pveg and SpecR-T in pSB1C3

Friday, August 20

Replica plating and colony PCR of both transformations from yesterday.

Annealing the forward and reverse strands of the dif P ES oligo

The forward and reverse strands of the 3' dif Pme1 sites 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.

Week 8

Day	Monday	Tuesday	Wednesday	Thursday	Friday
Morning	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
Afternoon	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

Week 9

Day	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 & 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

Week 10

Day	Monday	Tuesday	Wednesday	Thursday	Friday
Morning	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
Afternoon	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)

Week 11

Day	Monday	Tuesday	Wednesday	Thursday	Friday
Morning	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
Afternoon	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

Week 12

Day	Monday	Tuesday	Wednesday	Thursday	Friday
Morning	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	Start midiprep of C-Spec colonies again	Cloning digests of C-Spec with Kpn1 and Pst and N-Spec with Eco and Kpn1 Gel purification of the inserts
Afternoon	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!

Week 13

Day	Monday	Tuesday	Wednesday	Thursday	Friday
Morning	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	Start midiprep of C-Spec colonies again	Cloning digests of C-Spec with Kpn1 and Pst and N-Spec with Eco and Kpn1 Gel purification of the inserts
Afternoon	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!

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.

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

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