IGEM:Imperial/2010/Vectors team: Difference between revisions

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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.
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.


 
*[[IGEM:Imperial/2010/PyrD_Vector | Assembly of the<font color = #6B3FA0> '''PyrD vector''' </font>]]
* Assemble the<font color = #6B3FA0> PyrD vector </font>
*[[IGEM:Imperial/2010/AmyE_Vector | Assembly of the<font color = #CA2C92> '''AmyE vector'''</font>]]
* Assemble the<font color = #CA2C92> AmyE vector</font>


==Assembly of Vectors==
==Assembly of Vectors==
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[[Image:AmyE_assembly_Stategy.JPG |750px|thumb|center|alt=A|Overveiw of AmyE assembly]]
[[Image:AmyE_assembly_Stategy.JPG |750px|thumb|center|alt=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.
[[Image:K70_n_K64.JPG |250px|thumb|right|alt=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
[[Image:K02_and_oligos.JPG |250px|thumb|right|alt=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
[[Image:LacI_testing_construct |250px|thumb|right|alt=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==
===<font color = #6B3FA0>'''PyrD Vector'''</font>===
====Week 6====
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1"
|-
  ! Week 6 !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday
|-
  | MORNING ||  || ||  || ||
*<font color = #6B3FA0>Restriction digestion of 5' integration site for the PyrD vector ( Using Spe and Pst)
</font>
|-
  | AFTERNOON ||  || || ||
*<font color = #6B3FA0>'''Starting assembly of PyrD vector'''</font>
*<font color = #6B3FA0>Annealing the synthesized oligos ( Pme and diff sites)</font>
||
*<font color = #6B3FA0>Gel analysis of resultant products from digestion mixture</font>
*<font color = #6B3FA0>PCR purification of digestion mixture</font>
*<font color = #6B3FA0>Further gel analysis</font>
|}
====Week 7====
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1"
|-
  ! Week 6 !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday
|-
  | MORNING
||<font color = #CA2C92>'''Starting assembly of AmyE vector'''</font>
*<font color = #CA2C92>Restriction digestion of BB k14070 for the AmyE vector (using Eco and Spe)</font>
*<font color = #CA2C92>Restriction digestion of BB k14064 for the AmyE vector (using Eco and Xba)</font>
*<font color = #6B3FA0>Restriction digestion of 5' integration site (k08) for the PyrD vector (using Eco and Spe)</font>
*<font color = #6B3FA0>PCR amplification of vector backbone PSB1C3 for the PyrD vector</font>
||
*<font color = #6B3FA0>Gel analysis and extraction of 5' int site for PyrD vector (repetition of step due to absence of DNA during gel analysis yesterday)</font>
*<font color = #CA2C92>Gel purification of k70</font>
*<font color = #6B3FA0>Gel purification of k08</font>
*<font color = #CA2C92>Re-analysis of k70, k64 (for AmyE) on gel to work out ratios for ligation set up</font>
*<font color = #6B3FA0>Re-analysis of k08, Pme oligos and pSB1C3 (for PyrD) on gel to work out ratios for ligation set up</font>
*<font color = #6B3FA0>Restriction digestion of pveg promoter (k53) (using Eco and Spe) and spec cassette (k65) (using Xba and Pst) for PyrD vector</font>
*<font color = #6B3FA0>Restriction digestion of pSB1C3 for PyrD (using Eco and Pst)</font>
||
*<font color = #6B3FA0>Check for transformed colonies (colonies that have taken up the vector with the 5'diff) and prepare for colony PCR</font>
*<font color = #6B3FA0>Gel purification of k53 and k65 for AmyE in preparation for ligations this afternoon</font>
*<font color = #6B3FA0>Gel extraction and re-analysis on gel of k53, k65 and psB1C3 for Spec casette in preparation for ligations this afternoon</font> 
||
*<font color = #CA2C92>Transformation of overnight ligations of:
k64 and k70, k70 only, </font>
<font color = #6B3FA0>
Spec </font>
<font color = #6B3FA0>
and 5' PyrD diff</font>
||
*<font color = #6B3FA0>Replica plating and colony PCR of:
Spec and 5' PyrD diff</font>
*<font color = #CA2C92>Plate wash of:
k64 and k70. k70 only was discarded since this was purely for a baground check</font>
|-
  | AFTERNOON
||
*<font color = #6B3FA0>PCR purification of PSB1C3 vector </font>
*<font color = #CA2C92>PCR purification of BB k14064 digestion products</font>
*<font color = #CA2C92>Gel analysis of  digestion products of BB k14070</font>
*<font color = #CA2C92>Gel extraction of digestion products of BB k14070</font>
||
*<font color = #CA2C92>Restriction digestion of k64 and subsequent PCR purification (repetition of step due to absence of DNA during gel analysis)</font>
*<font color = #CA2C92>Gel analysis of k70, k64 (for AmyE) to work out ligation ratios</font>
*<font color = #6B3FA0>Gel analysis and extraction of k53 and k65</font>
*<font color = #6B3FA0>Bench (1  hour) and overnight ligation of 5'diff with the pSB1C3 vector</font>
*<font color = #6B3FA0>Transformation of E.Coli with the bench ligated vector</font>
||
*<font color = #CA2C92>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)</font>
*<font color = #6B3FA0>Set up overnight ligations of SpecR</font>
||
||
*<font color = #6B3FA0>Gel analysis of colony PCR products of Spec and 5' PyrD diff</font>
* Annealing of diff P oligos (used for both PyrD and AmyE vectors)
|}
===Week 6===
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1"
|-
  ! Week 6 !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday
|-
  | MORNING ||  || ||  || ||
*<font color = #6B3FA0>Restriction digestion of 5' integration site for the PyrD vector ( Using Spe and Pst)
</font>
|-
  | AFTERNOON ||  || || ||
*<font color = #6B3FA0>'''Starting assembly of PyrD vector'''</font>
*<font color = #6B3FA0>Annealing the synthesized oligos ( Pme and diff sites)</font>
||
*<font color = #6B3FA0>Gel analysis of resultant products from digestion mixture</font>
*<font color = #6B3FA0>PCR purification of digestion mixture</font>
*<font color = #6B3FA0>Further gel analysis</font>
|}
===Week 7===
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1"
|-
  ! Week 6 !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday
|-
  | MORNING
||<font color = #CA2C92>'''Starting assembly of AmyE vector'''</font>
*<font color = #CA2C92>Restriction digestion of BB k14070 for the AmyE vector (using Eco and Spe)</font>
*<font color = #CA2C92>Restriction digestion of BB k14064 for the AmyE vector (using Eco and Xba)</font>
*<font color = #6B3FA0>Restriction digestion of 5' integration site (k08) for the PyrD vector (using Eco and Spe)</font>
*<font color = #6B3FA0>PCR amplification of vector backbone PSB1C3 for the PyrD vector</font>
||
*<font color = #6B3FA0>Gel analysis and extraction of 5' int site for PyrD vector (repetition of step due to absence of DNA during gel analysis yesterday)</font>
*<font color = #CA2C92>Gel purification of k70</font>
*<font color = #6B3FA0>Gel purification of k08</font>
*<font color = #CA2C92>Re-analysis of k70, k64 (for AmyE) on gel to work out ratios for ligation set up</font>
*<font color = #6B3FA0>Re-analysis of k08, Pme oligos and pSB1C3 (for PyrD) on gel to work out ratios for ligation set up</font>
*<font color = #6B3FA0>Restriction digestion of pveg promoter (k53) (using Eco and Spe) and spec cassette (k65) (using Xba and Pst) for PyrD vector</font>
*<font color = #6B3FA0>Restriction digestion of pSB1C3 for PyrD (using Eco and Pst)</font>
||
*<font color = #6B3FA0>Check for transformed colonies (colonies that have taken up the vector with the 5'diff) and prepare for colony PCR</font>
*<font color = #6B3FA0>Gel purification of k53 and k65 for AmyE in preparation for ligations this afternoon</font>
*<font color = #6B3FA0>Gel extraction and re-analysis on gel of k53, k65 and psB1C3 for Spec casette in preparation for ligations this afternoon</font> 
||
*<font color = #CA2C92>Transformation of overnight ligations of:
k64 and k70, k70 only, </font>
<font color = #6B3FA0>
Spec </font>
<font color = #6B3FA0>
and 5' PyrD diff</font>
||
*<font color = #6B3FA0>Replica plating and colony PCR of:
Spec and 5' PyrD diff</font>
*<font color = #CA2C92>Plate wash of:
k64 and k70. k70 only was discarded since this was purely for a baground check</font>
|-
  | AFTERNOON
||
*<font color = #6B3FA0>PCR purification of PSB1C3 vector </font>
*<font color = #CA2C92>PCR purification of BB k14064 digestion products</font>
*<font color = #CA2C92>Gel analysis of  digestion products of BB k14070</font>
*<font color = #CA2C92>Gel extraction of digestion products of BB k14070</font>
||
*<font color = #CA2C92>Restriction digestion of k64 and subsequent PCR purification (repetition of step due to absence of DNA during gel analysis)</font>
*<font color = #CA2C92>Gel analysis of k70, k64 (for AmyE) to work out ligation ratios</font>
*<font color = #6B3FA0>Gel analysis and extraction of k53 and k65</font>
*<font color = #6B3FA0>Bench (1  hour) and overnight ligation of 5'diff with the pSB1C3 vector</font>
*<font color = #6B3FA0>Transformation of E.Coli with the bench ligated vector</font>
||
*<font color = #CA2C92>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)</font>
*<font color = #6B3FA0>Set up overnight ligations of SpecR</font>
||
||
*<font color = #6B3FA0>Gel analysis of colony PCR products of Spec and 5' PyrD diff</font>
* Annealing of diff P oligos (used for both PyrD and AmyE vectors)
|}
===Week 8===
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1"
|-
  ! Week 8 !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday !! Saturday
|-
  | MORNING
||
Set up overnight ligations for standard assembly (BBA) and 3A cloning (3A) of dif P
*<font color = #CA2C92>Restriction digestion of 3' integration site (K02) in the A2 vector ( Using Eco and Xba) for BBA
</font>
*<font color = #6B3FA0>Restriction digestion of 3' integration site (K09) in the AK3 vector ( Using Eco and Xba) for BBA
</font>
*<font color = #CA2C92>PCR purification of 3' integration site (K02) in the A2 vector ( Using Eco and Xba) for BBA
</font>
*<font color = #6B3FA0>PCR purification of 3' integration site (K09) in the AK3 vector ( Using Eco and Xba) for BBA
</font>
*<font color = #6B3FA0>Set up 5 ml culture of spec from colony 1 of replica plate in shaking incubator @ 37 degrees
</font>   
||
*<font color = #CA2C92>Restriction digestion of 3' integration site (K02) in the A2 vector ( Using Xba and Pst) for 3A
</font>
*<font color = #6B3FA0>Restriction digestion of 3' integration site (K09) in the AK3 vector ( Using Xba and Pst) for 3A
</font>
||
* 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.
*<font color = #CA2C92>Midiprep of CmR vector and test digest using Eco and Spe</font>
* Backbone PCR of PSB1C3 vector (1st attempt) for common use
 
||
*<font color = #6B3FA0>Replica plating of transformed colonies for k09 from the plate with the insert (diff P) - 45 sigle colonies were plated</font>
*<font color = #6B3FA0> The first 15 of the above colonies were colony PCRed using dif PES Fwd and pSB Rev</font>
||
*<font color = #6B3FA0>Gel analysis of colony PCR from yesterday (first 15 replica plated colonies</font>
*<font color = #6B3FA0>Set up further colony PCR reactions for the same 15 colonies using pSB Fwd and pSB Rev primers</font>
* Gel analysis of pSB1C3 - after backbone PCR and after digestion (looked contaminated!)
||
*<font color = #6B3FA0>Miniprep of 4 overnight cultures (dif P and 3' Insert for PyrD) - colonies 4,5,7 & 9</font>
|-
  | AFTERNOON
||
* Gel analysis of PCR purified K02 and K09 with the insert (diff P) in between to work out ratios for the liagation
*<font color = #CA2C92>Dephosphorylation of digested A2 vector with 3' integration site (K02)
</font>
*<font color = #6B3FA0>Dephosphorylation of digested AK3 vector with 3' integration site (K09)
</font>
*<font color = #CA2C92>Set up overnight ligation of A2 vector with 3' integration site (K02) with diff P (insert) and A2 vector only
</font>
*<font color = #6B3FA0>Set up overnight ligation of AK3 vector with 3' integration site (K09) with diff P (insert) and AK3 vector only</font>
*<font color = #6B3FA0>Colony PCR and gel analysis of plated culture (from plate wash on Friday) with K64 and K70</font>
*<font color = #6B3FA0>Overnight 100 ml culture of spec @ 14 degrees </font>
||
* 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
*<font color = #CA2C92>Set up overnight 100 ml culture of CmR vector (K64 and K70) for midiprep tomorrow</font>
* 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)
*<font color = #6B3FA0>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)</font>
||
*<font color = #6B3FA0>Diagnostic digests of minipreps - Two digests : One with Spe & Pst and other with Xba & Spe</font>
|}
===Week 9===
{| class="wikitable" style="text-align: center; width: 100%; height: 170px;" border="1"
|-
  ! Week 8 !! Monday !! Tuesday!! Wednesday !! Thursday !! Friday
|-
  | MORNING
||
*<font color = #6B3FA0>Gel analysis of Mini preps and diagnostic digests from Saturday - Colony 4 looked best</font>   
||
*<font color = #6B3FA0>Midiprep of Colony 4 - concentration 110 ng/ul</font>
||
*<font color = #6B3FA0>Gel purification of insert (35 ul)</font>
*<font color = #6B3FA0>Gel analysis of vector and insert to work out ratios for ligations</font>
||
*<font color = #6B3FA0> Transformation with overnight ligations</font>
||
*<font color = #6B3FA0> The transformations were highly successful!! The '''Vector only''' plate showed no colonies and the '''Insert & Vector''' plate showed many individual colonies</font>
*<font color = #6B3FA0> 10 individual colonies were replica plated and used for colony PCR</font>
||
|-
  | AFTERNOON
||
*<font color = #6B3FA0>Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow</font>
||
*<font color = #6B3FA0>Digests set up for Vector (SpecR) with Spe & Pst and Insert (dif P & K09)with Xba & Pst</font>
*<font color = #6B3FA0>PCR purification of vetor (35 ul)</font>
*<font color = #6B3FA0>Gel extraction of insert after gel analysis (35 ul)</font>
||
*<font color = #6B3FA0>Dephosphorylation of vector</font>
*<font color = #6B3FA0>Set up two overnight ligations; Vector & Insert and Vector only </font>
||
*<font color = #6B3FA0>Both ligations ('''Insert & Vector''' and '''Vector only''') were plated in CmR and incubated overnight</font>
||
*<font color = #6B3FA0>Gel analysis of colony PCR products</font>
|}
==Lab Notes==
===Thursday, 12 August===
===Friday, 13 August===
===Monday, 16 August===

Latest revision as of 12:30, 12 September 2010

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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.

Assembly of Vectors

A
Overveiw of PyrD assembly



A
Overveiw of AmyE assembly
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