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	==What We've Accomplished==		==What We've Accomplished==
-	[[Boston_UniversityPrimerDesign | Primer Design]]	+	[[Boston_UniversityPlasmidChoice | Plasmid Choice]]
-	[[Boston_UniversityRestrictionEnzymePlasmid | Plasmid and Restriction Enzyme Choice]]	+	[[Boston_UniversityRestrictionChoice | Restriction Enzyme Choice]]
-	== Week's (Ambitious) Goals ==	+	[[Boston_UniversityPrimerDesign | Primer Design]]
-	Wednesday 5/30	+	[[Boston_UniversityCompTransf | Making Shewanella Competent and Transforming Plasmid]]
-	# Get all protocols	+
-	#  Identify materials/prepare order	+
-	#  Design Primers	+
-	#  Learn about budget/POs	+
-		+
-	Thursday 5/31	+
-	# Do primer order	+
-	# Start conjugation practice	+
-	# Confirm restriction enzymes, ligases	+
-	# Order confirmed/needed materials	+
-	# Team Revew Meeting	+
-	# Draft Thank-You Letters for our Sponsors	+
-		+
-	Friday 6/1	+
-	# Evaluate/continue conjugation, practice electroporation for E. coli	+
-	# Revise proposal to include possibility of screening with alginate beads and fluorocytometer	+
-	# Meeting with Tim:  Budgets/protocols, Pfizer/fundraising, iGEM registration, beads	+
-		+
-	Week of 6/4:	+
-		+
-	1. Evaluate the transformation that was done on Friday.	+
-		+
-	2. Confirm the correct plasmid (pJQ200)	+
-		+
-	3. Find appropriate restriction enzymes that cut by BLASTing all the plasmid's restriction enzyme sites onto the global transcription factors.	+
-		+
-	4. Re-design primers for the global transcription factors based on the restriction enzymes we have selected.	+
-		+
-	5. Order primers.	+
-		+
-	6. Practice regular (non-error prone) PCR with the primers to check that they work.	+
-		+
-	7. Incorporate the global transcription factors into the plasmid and transform this plasmid into the E.coli.	+
-		+
-	8. Conjugate this plasmid into Shewy.	+
	== Materials We Need ==		== Materials We Need ==
-	Primers:  Need to Buy	+	Error-Prone PCR:  From CAB(?)
-	Error-Prone PCR:  Need to Buy	+	Ligases:  Need to Buy
-		+
-	Plasmids:  Need to Buy?	+
-		+
-	Restriction Enzymes:  Need to Buy?	+
-		+
-	Ligases:  Need to Buy?	+
	== Short-Term To-Do List ==		== Short-Term To-Do List ==
-		+	EDIT THE WIIIIKIIII
-	Lab Orientation: COMPLETED!	+
-	*[[Lab Orientation Checklist]]	+
-		+
-	Lab Safety Training: COMPLETED!	+
-		+
-	Design of Primers:  COMPLETED!	+
-		+
-	Ordering of Primers:  COMPLETED!	+
-		+
-	Gathering of Protocols:  Not Completed (Chris, please send me the protocols when they are gathered)	+
-		+
-	Ordering of Error-Prone PCR Materials:  Not completed	+
-		+
-	Thank-You Letters sent to Pfizer:  Not Completed	+
-		+
-	Thank-You Letters sent to BU ppl:  Not completed	+
	== Protocols ==		== Protocols ==
-	==="Calcium Chloride/Heat Shock Plasmid Transformations===	+	[[Boston_UniversityHeatShockProtocol | Calcium Chloride/Heat Shock Plasmid Transformations Protocol]]
-	Reagents to be Supplied by the User	+	[[Boston_UniversityFilterConjugationProtocol | Filter Cojugation Protocol]]
-	LB plates with appropriate antibiotic concentration (for pET -25b(+), amipicillin at 50-100 microgram/mL) SOC media	+	[[Boston_UniversityTOPO | pTrcHis TOPO TA Expression Kit Cloning Protocol]]
-	1) Prepare one LB-Amp plate for each transformation, plus one plate for a negative (no plasmid) control. After storage at 2-8 degrees C, equilibrate to room temperature.	+	[[Boston_UniversityNEBCutter| Using NEBCutter for checking specific restriction enzymes against a sequence]]
-	2) Centrifuge the tubes containing plasmid DNA to collect contents at the bottom of the tube. Add 1 microliter DNA to a sterile 1.5 mL tube on ice. Have one tube on ice with no DNA.	+	[[Boston_UniversityGel | Making an Electrophoresis Gel]]
-	3) Add 50 microliters of competent cells (either freshly prepared, or frozen and thawed on ice). Avoid excessive pipetting, as cells are very fragile.	+	[[Boston_UniversityZymo/SOB | Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB
-		+	]]
-	4) Gently flick the tubes to mix, and place on ice for 20 minutes.	+
-		+
-	5) Heat shock the cells for 45 seconds to 2 minutes in a water bath at exactly 42 degrees C. Do not shake.	+
-		+
-	6) Immediately return the tubes to ice for 2-10 minutes.	+
-		+
-	7) Add 950 microliters of room temperature SOC media to the tubes.	+
-		+
-	8) Incubate 1-1.5 hours at 37 degrees C with shaking (~150 rpm).	+
-		+
-	9) Plate 100 microliters of each transformation culture onto antibiotic plates.	+
-		+
-	10) Incubate the plates overnight (16-24 hours) at 37 degrees C.	+
-		+
-	See Making Heat Shock Competent Cells for more information. "	+
-		+
-	Negative Control:  No plasmid	+
-		+
-		+
-	'''Trial 1:'''	+
-		+
-	We followed this protocol from step 3 in order to practice our transformation and conjugation techniques. The competent cells given to us by Joshua were E.coli sm10; the GFP plasmids (pMS291/lacI) were supplied by Ilaria. But we performed the protocol with four samples of plasmid DNA+E.coli. Each sample contained 1 microliter of 63.5 ng/microliter GFP plasmid DNA; 50 microliters of the competent E.coli cells; and 950 microliters of SOC media. 100 microliters of each sample tube were plated onto one kanamycin plate and one blank (non-antibiotic), control plate.	+
-		+
-	'''Trial 1 results:'''	+
-		+
-	A lawn of bacteria on each plate. No GFP fluorescence under UV illumination.	+
-		+
-	'''Trial 2:'''	+
-		+
-	Used E.coli sm10 cells, GFP plasmid pMS291 with lacZ promoter (stimulated by IPTG).	+
-		+
-	Sample 1 contained 50 microliter sm10 + 1 microliter mPS291.	+
-		+
-	Sample 2 contained 50 microliter sm10 + 1 microliter water.	+
-		+
-	Sample 3 contained 50 microliter E.coli Dh5-alpha + 1 microliter water.	+
-		+
-	Samples 2 and 3 served as negative controls (should show no colonies in the presence of kanamycin).	+
-		+
-	5 microliters of each of these samples were plated onto [LB]+[20microliters of 50X kanamycin]+[20microliters of 100micromolar IPTG].	+
-		+
-	50 microliters of each of these samples was also plated onto [LB]+[20microliters of 50X kanamycin]+[20microliters of 100micromolar IPTG].	+
-		+
-	'''Trial 2 results:'''	+
-		+
-	Colony growth on all plates.  So the kanamycin is not killing any of the cells.  Perhaps the kan plates were made incorrectly.	+
-		+
-	'''Trial 3:'''	+
-		+
-	Repeated Trial 2 but with double volume of kanamycin and double volume of IPTG (to ensure stimulation of lacZ promoter an thus GFP expression).	+
-		+
-	'''Trial 3 results:'''	+
-		+
-	5 microliters sm10 + water in IPTG and kan: lawn of colonies	+
-		+
-	50 microliters sm10 + water in IPTG and kan: less than 10 colonies	+
-		+
-	5 microliters sm10 + pMS291 in IPTG and kan: less than 10 colonies, '''fluorescent under UV illumination !'''	+
-		+
-	50 microliters sm10 + pMS291 in IPTG and kan: less than 10 colonies, no fluorescence	+
-		+
-	5 microliters Dh5-alpha + water in IPTG and kan: more than 100 colonies	+
-		+
-	5 microliters sm10 + water in kan: lawn of colonies	+
-		+
-	5 microliters sm10 + pMS291 in kan: lawn of colonies	+
-		+
-	5 microliters Dh5-alpha + water in kan: more than 100 isolated colonies	+
-		+
-	E.coli successfully transformed with pMS291 (showed fluorescence).  These colonies will be grown out in SOC media  for further examination.  But kan still seems to be innefective.	+
-		+
-	[update] The succesfully transformed E.coli that fluoresced were grown out in SOC media and then centrifuged to obtain a pellet of cells.  This pellet definitely fluoresced under UV confirming that the transformation was succesful.	+
-		+
-	'''Trial 4:'''	+
-		+
-	Two stocks were made: one with 20 microliters E.coli Dh5-alpha + pMS291 (lacZ), and the second with 20 microliters E.coli Dh5-alpha + water.  Each of these two stocks were plated onto 4 different plates.	+
-		+
-	Plate 1 contained LB	+
-		+
-	Plate 2 contained LB + kanamycin	+
-		+
-	Plate 3 contained LB + kanamycin + IPTG	+
-		+
-	Plate 4 contained LB + kanamycin which was dripped onto the LB	+
-		+
-	'''Trial 4 Results:'''	+
-		+
-	''20 microliters E.coli Dh5-alpha + pMS291 (lacZ)''	+
-		+
-	Plate 1 containing LB: lawn of bacteria	+
-		+
-	Plate 2 containing LB + kanamycin: more than 100 colonies	+
-		+
-	Plate 3 containing LB + kanamycin + IPTG: more than 100 colonies and fluorescent!	+
-		+
-	Plate 4 containing LB + kanamycin which was dripped onto the LB: more than 100 colonies	+
-		+
-		+
-	''20 microliters E.coli Dh5-alpha + water''	+
-		+
-	Plate 1 contained LB: no colonies	+
-		+
-	Plate 2 contained LB + kanamycin: no colonies	+
-		+
-	Plate 3 contained LB + kanamycin + IPTG: no colonies	+
-		+
-	Plate 4 contained LB + kanamycin which was dripped onto the LB: no colonies	+
-		+
-	Therefore, the kanamycin worked correctly. E.coli without pMS291 (without kanamycin resistance) all died. Transformation was also successful	+
	== Question and Answer ==		== Question and Answer ==
-
-	== Relevant Publications and Links==
-	http://www.shewybase.bu.edu
	[https://2007.igem.org/Boston_University Back]		[https://2007.igem.org/Boston_University Back]

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Latest revision as of 16:43, 6 July 2007

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Contents 1 What We've Accomplished 2 Materials We Need 3 Short-Term To-Do List 4 Protocols 5 Question and Answer

What We've Accomplished

Plasmid Choice

Restriction Enzyme Choice

Primer Design

Making Shewanella Competent and Transforming Plasmid

Materials We Need

Error-Prone PCR: From CAB(?)

Ligases: Need to Buy

Short-Term To-Do List

EDIT THE WIIIIKIIII

Protocols

Calcium Chloride/Heat Shock Plasmid Transformations Protocol

Filter Cojugation Protocol

pTrcHis TOPO TA Expression Kit Cloning Protocol

Using NEBCutter for checking specific restriction enzymes against a sequence

Making an Electrophoresis Gel

Procedure for 50 mL E. Coli mixture in Zymo Broth or SOB

Question and Answer

Back