Construction

From 2006.igem.org

Revision as of 02:41, 18 September 2006 (view source) Chuck (Talk | contribs) ← Older edit		Revision as of 02:43, 18 September 2006 (view source) Chuck (Talk | contribs) Newer edit →
Line 1:		Line 1:
	== September 16, 2006 ==		== September 16, 2006 ==
		+	Charles:
		+	<ul>
		+	<li>Made 4 replications of UT2 in 200 uM, 100 uM, 50 uM, 25 uM and 0 uM of IPTG.
		+
		+	<li>Made frozen stock (2 vials each): UT01, I13507 (2005), B0015 (2005), B0031 (2005)
		+	<li>Mini-prepped UT2 ABCEDF, P0352 (2005) and P0152 (2005)
		+	</ul>
		+
		+	To-Do List:
		+	<ul>
		+	<li>Test the lengths of UT2 ABCEDF, P0352 (2005) and P0152 (2005)
		+	<ul>
		+	<li>Prepare an o/n growth of the “best” UT2 for testing
		+	<li>Prepare an o/n growth of regular DH5a-z1
		+	</ul>
		+	<li>Mini-prep UT03 ABCD and test their lengths along with P00352 (2005) and P0152 (2005)
		+	<li>Re-plate P0452 (2005) - AK, and P0456 (2005) - AC
		+	<li>Make 24 competent cells
		+	</ul>
	== September 15, 2006 ==		== September 15, 2006 ==

---

Revision as of 02:43, 18 September 2006

Contents 1 September 16, 2006 2 September 15, 2006 3 September 14, 2006 4 September 13, 2006 5 September 12, 2006 6 September 11, 2006 7 September 10, 2006 8 September 9, 2006 9 September 8, 2006 10 September 7, 2006 11 September 6, 2006 12 September 5, 2006 13 September 4, 2006 14 September 3, 2006 15 September 2, 2006 16 September 1, 2006 17 August, 11, 2006

September 16, 2006

Charles:

• Made 4 replications of UT2 in 200 uM, 100 uM, 50 uM, 25 uM and 0 uM of IPTG.
• Made frozen stock (2 vials each): UT01, I13507 (2005), B0015 (2005), B0031 (2005)
• Mini-prepped UT2 ABCEDF, P0352 (2005) and P0152 (2005)

To-Do List:

• Test the lengths of UT2 ABCEDF, P0352 (2005) and P0152 (2005)
  • Prepare an o/n growth of the “best” UT2 for testing
  • Prepare an o/n growth of regular DH5a-z1
• Mini-prep UT03 ABCD and test their lengths along with P00352 (2005) and P0152 (2005)
• Re-plate P0452 (2005) - AK, and P0456 (2005) - AC
• Make 24 competent cells

September 15, 2006

Andy, Charles, Natalie, Stan, Elliott:

• Checked and transformed I+J+I plasmids (Bands: ~6000-5000, 3500-2500 (smear))
• Made competent cells (although, may have to repeat)
• Prepared o/n growths of I+J+E (12 vials), P0152 (2005) and P0352 (2005). P0456 (2005) and P0452 (2005) did not seem to grow very well so left them in the incubator for a 2nd night
• Made 8 Amp plates

To-Do List:

• Mini-prep P0152 (2005), P0352 (2005) and 6 of the I+J+E
• Try to get J04450 (2006) working again.
• Check lengths of R0011 (2005/6), if no good, then obtain R0010 (2005/6) from Regsitry
• Make freezer stock (2 each) of I+J, B0015 (2005), B0031 (2005), and I13507 (2005) and put in -80C freezer stock
• Test the fluorescence of I+J+E

September 14, 2006

Andy, Natalie:

Part Name	Parts Match	Plasmid Match
I0500 E (2005)	--	O
I0500 F (2005)	--	O
I+J+E AA-CB (2005)	O	O

• all the I+J+E bands were roughly in the right location (base pair bracket) ~60% confidence
• ligated I+J with I13507 (made 6 replicates)

Andy, Charles:

• Made o/n of cells to be made competent
• Transformed I+J+E into 6 plates, P0456 (2005), P0152 (2005), P0352 (2005), and P0452 (2005).
• Made plates: 10 Amp, 1 Amp/Cam, 3 Amp/Kan

To-Do List:

• Make o/n growth of P0456 (2005), P0152 (2005), P0352 (2005), and P0452 (2005).
• If I+J+E colonies formed, make o/n growths at the 2 extreme temperatures and 1 at 37C
• Transform/check I+J+I plasmids.
• Make 6 Amp plates
• Make 30 competent cells
• Make o/n growth of I+J, B0015 (2005), B0031 (2005), and I13507 (2005) to make -80C freezer stock

September 13, 2006

Digested I13507 (2005) and extracted plasmid. B = 15 ng/uL, C = 30 uL (had an extra band at 1500…so ignored it and hopefully we got the right part at ~3000)

Ligate I+J ABC with E0240 AB (2005) in all the combinations: AA, AB, BA, BB, CA, CB (part – plasmid)

To-Do List:

• Redo digestion/ligation/quantitation page
• Make more plates: Amp = 7, Amp/Kan = 2, Amp/Cyc = 1
• get following parts from the 2005 Registry: P0456 (2005) – 16 O, pSB1AC3; P0152 (2005) – 19 I, pSB1A2; P0352 (2005) – 21 E, pSB1AK3; P0452 (2005) – 23 A, pSB1AK3 and transform cells
• Transform cells with I+J+E (all 6)

September 12, 2006

Andy, Anne, Charles, Natalie:

• Mini-prepped I+J HIJK
• Checked ABCDEFG but only ABCD worked, b/c EFG had thee bands – try again later?
• Quantitated I+J: A = 25 ng/uL; B = 15 ng/uL; C = 20 ng/uL

Andy, Elliott, Ting:

• Mini-prepped B0015 (2006) x 2, I0500 (2005) + J06801 (2006) 1:2 x 1, I0500 (2005) + J06801 (2006) 1:3 x 2.
• One I0500 (2005) + J06801 (2006) 1:2 overnight growth had no cells.

To-Do List:

• Make more plates: Amp = 7, Amp/Kan = 2, Amp/Cyc = 1
• cut I13507 (2005) with EcoRI and XbaI with Buffer 2
• get following parts from the 2005 Registry: P0456 (2005) – 16 O, pSB1AC3; P0152 (2005) – 19 I, pSB1A2; P0352 (2005) – 21 E, pSB1AK3; P0452 (2005) – 23 A, pSB1AK3 and transform cells
• ligate I+J with E0240 (2005) at a 1:3 = plasmid : insert molar ratio. And double digest to check the lengths, meanwhile, plate the cells.

September 11, 2006

Made Mini-preps of B0015 (2005) – need to test tomorrow – and made o/n of B0015 (2005) for a freezer stock. B0015 (2006) didn’t seem to grow very well so let it grow for another day and mini-prep tomorrow.

Made many (at least 6) o/n growths of the new part I0500 + J06801

To-Do List:

• mini-prep B0015 (2006) – if cells have grown
• mini-prep all the I0500 + J06801 vials. Label them I+J
• test the mini-preps for correct lengths (I+J will have a plasmid length of pSB2K3 and a part length of 1210 + 1371 = 2581 bp long)
• if test is good, proceed to ligation with E0240 (2005) and I13507 (2005). – NOTE: need to digest the plasmid (E0240 and I13507) with EcoRI and XbaI and to digest the insert (I+J) with EcoRI and SpeI – this is a pre-insertion.
• Once ligation is complete, do an initial check to see if ligation was successful, if so, transform cells with the new parts I+J+E and I+J+I.

September 10, 2006

Miniprepped a lot of parts:

Part Name	Parts Match	Plasmid Match
I0500 C (2005)	X	O
I0500 D (2005)	X	O
E0240 C (2005)	O	O
E0240 D (2005)	O	O
B0031 C (2005)	--	O
B0031 B (2005)	--	O
I12006 D (2005)	X	X
I12006 C (2005)	X	X
I13507 B (2005)	O	O
I13507 C (2005)	O	O
J06801 D (2006)	O	O
J06801 C (2006)	O	O
C0056 B (2005)	X	O
C0056 C (2005)	X	O
B0034 B (2005)	--	O
B0034 C (2005)	--	O

Made an o/n growth of B0015 (2005/2006) to have miniprepped and to make freezer stock

LOOKS LIKE LIGATION WORKED!!! The initial check showed that there was a band at ~4500 (plasmid), a band at ~2000 (new part) and a band at ~1000 (old part). So, cells were transformed with the new part and plated. It seems like 1:2 molar ratio of plasmid to insert worked the best (brightest new part and faintest old part)

To-Do List:

• miniprep B0015 (2005/6) and I0500 (2005)
• prepare o/n growth of new part

September 9, 2006

Mini-prepped everything (from yesterday) Plated B0015 (2005) and B0015 (2006) Decided to have a larger stock of mini-preps so made double amounts of o/n growth of I0500 (2005), E0240 (2005), B0031 (2005), I12006 (2005), I13507 (2006), J06801 (2006), C0056 (2005), B0034 (2005)

September 8, 2006

Double digested parts B0015 (2005), C0056 (2005) and I13507 (2005) again with EcoRI and PstI with Buffer EcoRI. To ligate the parts, I0500 (2005) will be the host and J06801 (2006) will be the insert. The new part will then be then inserted into the E0240 (2005) host. Therefore, I0500 will be cut with SpeI/PstI, J06801 will be cut with XbaI/PstI, and E0240 will be cut with EcoRI/XbaI.

Part Name	Parts Match	Plasmid Match
B0015 A (2005)	O	O
C0056 A (2005)	X	O
I13507 A (2005)	O	O

Check c0056 A (2005) again

The quantitation results are as follows:

• E0240 B, 8.0 ng/uL
• E0240 A, 6.0 ng/uL
• I0500 B, 13.5 ng/uL

To-Do List:

• transform cells with B0015 (2005)
• MP B0031 (2005), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005), I13507 (2005), J06801 (2006)
• Ligate I0500 + J06801

September 7, 2006

Obtained purified DNA of I0500 (2005) with a concentration of 0 ng/uL. Try again tomorrow

Made mini-preps of B0015 (2005) and I13507 (2005) and made a plate of B0031 (2005)

Part Name	Part Description	Parts Match	Plasmid Match
B0015 (2005)	Terminator	--	--
B0031 (2005)	Medium RBS	--	O
C0056 (2005)	Repressor protein 434 cI	--	O
I13507 (2005)	RBS.mRFP.Terminator	--	--

Do it all again except B0031 (2005) because part length ~14 bp. C0056 ~ 800 bp when it should be 636 bp and I13507 had really fat bands.

To-Do List:

• Purify I0500 (2005) at a higher concentrating (X3) (tape wells together)
• Purify E0240 (2005) while testing part lengths of B0015 (2005), C0056 (2005) and I13507 (2005) again
• Check compatibilities with cI plasmids and I12006 + E0240
• Ligate Parts I0500 (2005), J06801 (2006), E0240 (2005)

September 6, 2006

We started the mini-prep procedure for B0015 (2005) and I13507 (2005) by preparing an o/n growth of cells. We then prepared freezer stock of J06801 (2006), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005).

We began the ligation procedure using I0500 (2005) and J06801 (2006). We were only able to get J06801 to work so we proceeded with the quantitation of that part only. We obtained 3.0 ng/uL of purified J06801 DNA ready to be ligated tomorrow.

To-Do List:

• Repeat ligation attempt using I0500 (2005) and maybe continue with E0240 (2005)
• If plasmids are compatible, we can ligate I12006 (2005) and E0240 (2004) and co-transform with a plasmid that expresses cI
• Transform B0031 (2005) to make a freezer stock after 2 days.
• Complete MP procedure for B0015 (2005), I13507 (2005)
• Then check part/plasmid length, (with double digest), of B0031 (2005), C0056 (2005), B0015 (2005), I13507 (2005)

September 5, 2006

We tested the newly prepared MPs as well as two of the parts from yesterday using a double digest (XbaI and SpeI with Buffer 2).

Part Name	Part Description	Parts Match	Plasmid Match
J06801 a (2006)	Inverter with strong RBS	O	O
J06801 b (2006)	Inverter with strong RBS	O	O
J06501 (2006)	Repressor, LacI temp sensitive	X	X
B0034 (2005)	RBS (Elowitz 1999)	--	O
C0056 (2005)	Repressor protein 434 cI	--	O
E0240 (2005)	Medium RBS.GFP.Terminator	O	O
E0422 (2005)	RBS.ECFP.Terminator	X	X

We transformed B0015 (2005) and I13507 (2005).

To prepare a freezer stock, we made o/n of J06801 (2006), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005)

It now looks like we can attempt a ligation according to the first stage in the proposed CDR modules (I0500 + J06801 + E0240).

To-Do List for Tomorrow:

• MP B0015 (2005), I13507 (2005)
• Make a freezer stock of 06801 (2006), B0034 (2005), C0056 (2005), E0240 (2005), I0500 (2005), I12006 (2005)
• Ligate I0500 (2005), J06801 (2006), and E0240 (2005)

September 4, 2006

Started the mini-prep (MP) procedure (o/n growth) on E0240 (2005), B0034 (2005), J06801 (2006) and J06501 (2005)

Obtained a MP for C0056 (2005) and E0422 (2005).

Plan to do:

• Check lengths of E0422 (2005) and C0056 (2005)
• Transform B0015 (2005) as a backup/practice
• MP B0034 (2005), E0240 (2005), J06501 (2006), and J06801 (2006) -- check lengths once done
• Make I13507 (2005/2006) -- RFP (minus a promoter)

September 3, 2006

We decided to recheck the more relevant parts using a double digest (XbaI and SpeI with Buffer 2). This time we compared both the plasmid length and the actual part length. Again O = match, X = no match and -- = inconclusive)

Part Name	Part Description	Parts Match	Plasmid Match
I0500a (2005)	Promoter inducible by pBad/araC – part of thermometer	O	O
I0500b (2005)	Promoter inducible by pBad/araC – part of thermometer	O	O
J06801 (2006)	Inverter with strong RBS	O	O
B0031 (2005)	Medium RBS	--	O
B0031 (2006)	Medium RBS	X	X
C0056 (2006)	DNA sequence for repressor protein 434 cI	X	X
B0015 (2006)	Terminator	--	O
J04450 (2006)	RFP switched off by IPTG	X	X
I12006 (2005)	Modified lamdba Prm promoter (repressed by 434 cI)	--	O
E0240 (2006)	Medium RBS.GFP.Terminator	X	X

Began min-prep procedure (o/n growth) for C0056 (2005) and E0422 (2005).

Transformed new parts from the 2005 DNA plate, (E0240, B0034, and J06501), and transformed more of the J06501 (2006) from the stock solution to produce more DNA for the mini-prep.

September 2, 2006

Now that we knew the enzymes are working, we proceeded to test the MiniPreps from 2005 and 2006 (O = match, X = no match):

	Part Name	Part Description	Parts+Length Match
Gel 1	C0051 (2005)	Repressor, Lambda cI	X
	E00433 (2005)	Reporter containing LacZ alpha fragment	O
	B0031 (2005)	Medium RBS	O
	R0040 (2005)	Promoter driven by tetR but action inhibited by aTc	O
	R0011 (2005)	Promoter regulated by lacI – part of BBa_J06801 (Inverter)	O
Gel 2	I0500 a (2005)	Promoter inducible by pBad/araC – part of thermometer	O
	I0500 b (2005)	Promoter inducible by pBad/araC – part of thermometer	O
	B0031 (2006)	Medium RBS	X
	C0056 (2006)	DNA sequence for repressor protein 434 cI	X
	E0422 (2006)	RBS.ECFP.Terminator	X
	J04450 (2006)	RFP switched off by IPTG	X
	E0240 (2006)	Medium RBS.GFP.Terminator	X
	R0011 (2006)	Promoter regulated by lacI – part of BBa_J06801 (Inverter)	X
	E0433 (2006)	Reporter containing LacZ alpha fragment	O

We also began the transformation of some of the 2005 parts:

• I0500 (Promoter inducible by pBad/araC – part of thermometer)
• C0056 (DNA sequence for repressor protein 434 cI)
• I12006 (Modified lamda Prm promoter repressed by 434 cI)
• E0422 (RBS.ECFP.Terminator)

September 1, 2006

With a lot (a LOT) of help from Seema, we now have 24 Eppendorf tubes of competent cells (DH5a-z1) in the -80C freezer. We also have ~10 plates with Ampicillin (Amp), ~8 plates with Kanamycin (Kan), and 4 plates with Amp/Kan resistance. Seema also sorted out our iGEM box and separated between the 2005 parts and the 2006 parts as well as grouped the enzymes, reagents, and ladders.

Due to a lot of problems with previous attempts at transformations and quantifications, we decided to begin testing the enzymes as well all the part lengths so that we can get a handle of what we have to work with. So we tested all the enzymes, (EcoRI, XbaI, SpeI, and PstI) using a test plasmid. In theory, the uncut plasmid should move farther in the gel than the cut plasmid because the circular plasmid is more compact and would better be able to move through the agarose gel. This was indeed the case and all the enzymes cut successfully as shown the by the 4 bands being at the same height above the uncut band.

One thing to note was that the enzyme Xba1 worked slower than the others. Since the idea was to just check if the enzymes were working, we didn’t need to let the enzymes cut for the full 1 hr (as per the protocol). Thus, we ran the gel about 40 min early and saw two bands (one for each of the cut and uncut plasmid) in the Xba1 lane.

August, 11, 2006

Hey team,

It looks like we'll soon be underway and get to start building and testing parts. I seem to remember Farshid saying he and Jessica already extracted the parts that we needed from the registry, so that we can move on from there. There's a semi-final outline of what we'd like to do on the Critical Design Review (CDR) page so take a look at that. Once we get approval from Prof. Davies, we can get started.

The overall idea is to "assign" a part to a member so that member is responsible for growing/maintaining that part as well as adding that part to someone else's part. Also, depending on other people's thoughts on this, it would be a good idea to double up on the parts so that we get at least two cracks at making a part at the same time. This allows everyone to participate in the process from start to finish. Of course there will be a lot of flexibility in who does what, but this gives us some sort of baseline to start with. I'll be making some sort of sign-up sheet so if you want to make a specific part for some reason you can. I was thinking of using Google calendar to help with the coordination, so if you want a Gmail address, you can just email me and I can invite you (Gmail is actually really good). I'll keep you updated on this idea though.

Finally, CHECK THE WIKI REGULARLY!! We are now going to start doing stuff so there should be regular postings on the Wiki and so I don't want to flood everyone's inboxes. I will try to see if there is some way to indicate whether a link has been modified in some way so that you know there has been an update. Or if any of you know how to implement that by all means go for it.

Charles Yoon

[http://2006.igem.org/University_of_Toronto_2006 Home]