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
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
Next Steps
- Purify the correct insert out of E.coli
- 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
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 5' Ins ( synthesized oligos )
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.
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 digested vector.
PCR purification of the digested dif XP
The digested 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
Week 6
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 digest of pveg
Wednesday, August 18
Thursday, August 19
Friday, August 20
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