Berk2010-Amy

June entries

To Do

Background stuff:

• Plan self-lysis timing tests (see Jin's email)
• look up eGFP expression in other single celled eukaryotes. Are we using a GFP that could even express in Choanos?
• look up transposase expression times
• reverse transcriptase--deliver rRNA
• integrase... needs integration

Amy N. Kristofferson 13:04, 21 July 2010 (EDT)

To do:

• Sequence analysis
• Next step of assembly for atub and ef1a (despite possible deletion)
  • One-pot reaction w/ 2093
• Next step of assembly for whatever sequenced properly
• See if i have any ef1a MPs that mapped well that I could submit for sequencing. I want one w/o the double T deletion. However, it's prob before the atg used as a start codon, so it may not affect anything. We'll see...
• On a second glance, mapping data for ig114/2271+2017 could be ok. Maybe do with iGEM10_043/044 (four reactions total) and submit them for sequencing?

Assembly of iGEM10_018 and 017

Manual R/L Digest of iGEM10_018 and 017. Started at 10:00am. Zymo'd. Ligation started at 1:08pm. Transform into Righty cells.

One Pot Assembly of atub/ef1a with jh2093

• Set up the digestion:

1uL lefty plasmid(methylated)
1uL righty plasmid (methylated)
1uL NEB2+ATP
0.3 uL XhoI
0.3 uL BglII
0.3 uL BamHI
6.1uL Water

• Digest for 1 hour at 37C
• Heat kill at 65 for 20 min
• Add 0.3uL T4 Ligase
• Ligate at room temperature for 30 minutes
• Put on ice for transformation
• Transform, rescue, plate on dual antibiotic plates

Amy N. Kristofferson 13:45, 20 July 2010 (EDT)

None of the colonies picked yesterday from the iGEM10_047, 48, 51, 52, or 16+2294 were co-transformed. And since the ColPCR didn't reveal much of anything, I'm just going to miniprep the first two samples of each, except for iGEM10_047. iGEM10_047's d sample looked great on ColPCR, so I'll miniprep sample a and d instead of a and b. Also, somehow all 12 of the colonies that I picked for the transfer of 2271/ig114+ 2017 from the hybrid into PMLL4AC seemed to still be in the hybrid vector, based on my analytical pcr (they all had bands around 900bp). I'll miniprep four of each anyway and map them. If they're not the right side, I'll do the transfer over again.

To do:

• Minipreps
• Ask Tim about Ef1a sequence.

Picking colonies w/ Payload and Payload delivery

30µL into ea mL TB media (use TB for growing up for assay)

Miniprep Map

Tube	Mapping	iGEM name	Digest	Expected length	Expected length if still in hybrid	Good?
1,2	2271+2017 w/ PMLL4AC	iGEM10_042	Eco/XhoI	3768, 1672	4702, 1672	Possibly. Upper band looks good. Perhaps lower band too faint
3,4	ig114+2017 g.p. a w/ PMLL4AC	iGEM10_041	Eco/XhoI	3998, 1672	4932, 1672	Possibly. Upper band looks good. Perhaps lower band too faint
5,6	2345 in PMLL4 b w/ 2114+2316 g.p. a	iGEM10_047	Eco/Bam	5533, 2877		a,b
7,8	2345 in PMLL4 b w/ 2114+2275 a (7/14)	iGEM10_048	Eco/Bam	6102, 2877		a,b*
9,10	1968 in PMLL4 b w/ 2114+2316 g.p. a	iGEM10_051	Eco/Bam	5763, 2877		a,b
11,12	1968 in PMLL4 b w/ 2114+2275 a (7/14)	iGEM10_052	Eco/Bam	6332, 2877		a.b
13,14	16a w/ 2294		Eco/Bam	3166, 2745	righty methylated!!	good for one cut, but need to do Eco/XhoI

Submitted a sample a for each of the 2345/1968 assemblies for sequencing, except I submitted sample d for iGEM10_047.

16+2294 is righty methylated. That's why I only got one band. It's the right sized band though. I'll map it with Eco/XhoI. Started digest at 4:10pm.

Lane 1: 16+SLa
Lane 2: 16+SLb

Expected lengths: 4644, 1466

Analysis: I think the top band is parent vector. Don't know why sample B has three bands... very strange. I submitted a for sequencing.

Redo of Hybrid vector transfer

Those oligos shouldn't PCR off the genome (http://archaea.ucsc.edu/cgi-bin/hgPcr?hgsid=462798&org=Escherichia+coli+K12&db=eschColi_K12&wp_target=genome&wp_f=cggttatccacagaatcaggg+&wp_r=gcgctgatgtccggcggtgc&Submit=submit&wp_size=8000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0)

I'm going to make more of pMLL4AC Eco/Bam digest from Tim's stock in plate 2, well F7 (pMLL4AC Bth8062). Started digestion at 2:04pm. Drop out is 813bp. Vector should be 2745bp.

Use 3uL of digested pMLL4AC instead of 1uL when ligating.

Unfortunately, there's only about 1.75uL of digest PMLL4AC vector left, so I used it to redo the ligation w/ eco/bam digested ig114+2017. I started the ligation at 1:51pm. Started rescue at 3:04pm.

Redo of ligation with new pre-digested and gel purified PMLL4-AC w/ FFGFP dropout. Started ligation at 4:45pm. Started rescue at 5:30pm.

Amy N. Kristofferson 14:51, 19 July 2010 (EDT)

Everything grew up very well. The only concern I have is that the colonies with 2345 in the part have a transparent loop around the colony, possibly suggesting that self-lysis is starting. I'll have to careful when I miniprep these.

I pick colonies, ran a normal colony pcr for all of them, ran an analytical pcr to make sure the parts I transfered out of the hybrid vector aren't still in the hybrid vector, inoculated and check for co-transformation for the assembly plates. Here are my notes:

• pick four colonies off each plate.
• run colPCRs w/ ca998/g00101 for all of them
• run colPCRs w/ th395/th396 on ig114+2017 and 2271+2017 vector transfers and original minipreps to check for hybrid

Timing wise:

• make colPCR MM for 5 rxns w/ th395/396.
• set up tubes for colPCR (need 28 tubes for regular, and 8+2=10 for th395/396 version)
• set up plate for co-transformation check: 5x4 grid
• cross out gfp colonies on ig114/2271+2017 plates
• set up two 24-well plates. Need 2AC-4AK-1AC LB.
• pick colonies, colPCR (don't forget to double dip for the 2017s!), inoculate, co-transf. plate
• don't forget to add template DNA from original minipreps for th395/6 PCR for comparison check.

Analytical PCR results for Transfer from hybrid vector

This is a gel from a colPCR with oligos th395/th396 of four colonies picked from 2271+2017 and ig114 7/16/10 plates. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. This gel suggests that all the colonies I picked contained the hybrid vector. Tim said that if I need to do the transfer over again, I should use more pMLL vector (~3uL). For now, I'll pick more colonies.

Tube	Part
1a-4a	iGEM10_042
5a-8a	iGEM10_041
9a	2271+2017 in hybrid vector
10a	ig114+2017 g.p. in hybrid vector

ColPCR

This results aren't very good. But I think that's to be expected when parts are about 6k or greater. I'm going to mostly ignore these results and miniprep a couple samples from each reaction anyway.

The analytical PCR makes it seem like all the hybrid transfers are still in the hybrid vector... :(

ColPCR tube	DNA	iGEM name	oligos	Expected length	Good?
1--4	2271+2017 w/ PMLL4AC	iGEM10_042	ca998/g00101	2895
5--8	ig114+2017 g.p. a w/ PMLL4AC	iGEM10_041	ca998/g00101	3126
9--12	2345 in PMLL4 b w/ 2114+2316 g.p. a	iGEM10_047	ca998/g00101	5733	d
13--16	2345 in PMLL4 b w/ 2114+2275 a (7/14)	iGEM10_048	ca998/g00101	6302
17--20	1968 in PMLL4 b w/ 2114+2316 g.p. a	iGEM10_051	ca998/g00101	5963
21--24	1968 in PMLL4 b w/ 2114+2275 a (7/14)	iGEM10_052	ca998/g00101	6532
25--28	16a w/ 2294		ca998/g00101	3366
29--36	2271+2017 w/ PMLL4AC		th395/396
Small gel: 1--8	ig114+2017 g.p. a w/ PMLL4AC		th395/396

Amy N. Kristofferson 15:20, 16 July 2010 (EDT)

To do:

-Eco/Bam digest ig114+2017 g.p. and 2271+2017 g.p. Run on gel and gel purify. Ligate to pMLLAC pre-digested vector. (calculated expected lengths before running gel, to make sure it's worth the trouble. Bands might be so similar that maybe just a zymo is better)

-ligate g.p. 2294 with g.p. 16a.

-assemble SL-L with vacuole buster:

• Bjh2345 in pMLL4-AC with iGEM10_043 and iGEM10_044 (I'm going to use my 2114+2275 miniprep from 7/13, even though it hasn't been sequenced yet. It mapped well, but this doesn't say much since Pcon is so small. Even so, I'm do the reaction anyway and trash it if the sequence comes back bad, like the last one did.)
• Bjh1968 in pMLL4-AC with iGEM10_043 and iGEM10_044

NOTE: can't use one pot because iGEM10_043 and iGEM10_044 are NOT methylated. I transformed jtk030 cells to make them. I'll have to do separate righty and lefty digests.

Sequence Analysis of Ef1a/Atub

iGEM57-ef1a	ef1a animal promoter (iGEM10_021 in PMLLKA) miniprepped 7/14		ca998	Deletion of two T\'s at around 410bp. Would cause a frame shift, so it\'s useless.
iGEM58	atub animal promoter (jh2342 in pMLLKA) miniprepped 7/14		ca998	Perfect partial. The read was very short. Only the first 200bp or so were good.

So where to go from here?

I'll submit atub for sequencing with g00101. As for ef1a, since my ef1a in the AK vector (was supposed to be KA... argh) sequenced perfectly. I'll just Eco/Bam Transfer it? Then I'll get colonies tomorrow, and I can ColPCR/inoculate and then miniprep on Tuesday. OR I could pick more colonies today. However, even if I map well, the part could have mutations...

Transfer of 2271/ig114+2017 to PMLLAC from pMLL/1601 hybrid

Eco/Bam digest of 2271+2017 g.p. a, ig114+2017 g.p. a.

Gel purify part:

2271+2017 in pMLL/1601 vector Eco/Bam: 3670, 2704 Lower band includes parts.

ig114+2017 in pMLL/1601 vector Eco/Bam: 3670, 2934 Lower band includes parts

When I pick colonies, I'll run two ColPCRs: ca998/g00101 and th395/th396. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. I'll also run the ColPCR on colonies that I know contain the hybrid so that I can compare. This will allow me to do less minipreps. I'll still map them though, to confirm the vector size.

TH395   gcataGAATTCcggttatccacagaatcaggg        fwd for EcoBAM p15A, pBth7011
TH396   ctaggGGATCCgcaccgccggacatcagcgc         rev for EcoBam p15A, pBth7011

Second Step of Assembly for SL/VB: L-1968/2345 to iGEM10_043/044

Bam/XhoI: 2345 in PMLL4 b, 1968 in PMLL4AC (T1, L) Bgl/XhoI: 2114+2316 g.p. a, 2114+2275 a (7/14)

Details of today's Digestions, Ligations, Transformations, and Platings

Tube	DNA	Enzymes
1	2271+2017 g.p. a	Eco/Bam
2	ig114+2017 g.p. a	Eco/Bam
3	2345 in PMLL4 b	L: Bam/XhoI
4	1968 in PMLL4AC (T1, L)	L: Bam/XhoI
5	2114+2316 g.p. a	R: Bgl/XhoI
6	2114+2275 a (7/14)	R: Bgl/XhoI

Tube	DNA	Transform into	Plate on
1	2271+2017 w/ PMLL4AC	Lefty	AC
2	ig114+2017 g.p. a w/ PMLL4AC	Lefty	AC
3	2345 in PMLL4 b w/ 2114+2316 g.p. a	jtk030	AK
4	2345 in PMLL4 b w/ 2114+2275 a (7/14)	jtk030	AK
5	1968 in PMLL4 b w/ 2114+2316 g.p. a	jtk030	AK
6	1968 in PMLL4 b w/ 2114+2275 a (7/14)	jtk030	AK
7	16a w/ 2294	Righty	CA

Submit 2114+2275a 7/13 miniprep for sequencing

Submissions:

iGEM60	2114+2316a 7/13 miniprep fwd		ca998
iGEM61	2114+2316a 7/13 miniprep rev		g00101

Results: Great! This part is as it should be.

Sequencing Results for ig114+2017

iGEM59	ig114+2017 g.p.	ca56	Perfect partial: shows the correct junction between Pbad and SL-T. These three reads don\'t all overlap, but I think it\'s safe to assume the part is Perfect.

I'm going to Eco/Bam Transfer the part, because it's currently in a pMLL-jh1601 fusion backbone.

Amy N. Kristofferson 14:55, 15 July 2010 (EDT)

To do:

• reinterpret mapping results w/ SL with 1601 plasmid
• Design oligo for sequencing of 2271+2017
• gel purify SL. Ligate to gp'd 16a
• sequence analysis of ef1a and atub

Mapping Yesterday's Minipreps

Ran out the gel a bit more, and I think it shows that the two 2271+2017 samples mapped well:

Lane	Miniprep	Digest	Expected Lengths	'	Cut out	Good?
1	2114+2275a	Eco/XhoI	4627, 1466			good
2	2114+2275b	Eco/XhoI	4627, 1466			good
3	2271+2017a	Eco/XhoI	3768, 1672			too big
4	2271+2017b	Eco/XhoI	3768, 1672			too big
5	ig114+2017e	Eco/XhoI	3998, 1672			too big
6	ig114+2017f	Eco/XhoI	3998, 1672			too big
7	ef1a in PMLLKA b	Eco/Bam	2868, 897			good
8	atub in PMLLKA b	Eco/XhoI	2405, 1598			good
9	atub in PMLLKA c	Eco/XhoI	2405, 1599			bad
10	jh2294 in PMLLKA	BglII/XhoI	3777, 1607		Upper band	good

Sequencing Results for the rest of the Minipreps from my first batch of colony picks from Gel-Purified Assembly

Submission Name	Part	Oligo	Analysis
iGEM51	2114+2275 g.p. F	ca998	BAD- a bunch of junk. No alignment with sequence.
iGEM52	2114+2275 g.p. R	g00101	Perfect partial. The last 1000bp or so of the part is perfect
iGEM53	2271+2017 g.p. F	ca998	Perfect Partial- first ~1000bps of the part is right
iGEM54	2271+2017 g.p. R	g00101	Perfect Partial- last ~700bp of part look good
iGEM55	ig114+2017 g.p. F	ca998	About 1000bp aligned, except for mutation: @ 291bp, mutation: @ 449bp
iGEM56	ig114+2017 g.p. R	g00101	Perfect Partial: last 700bp sequenced perfectly

The boundaries of parts 2114+2275 and ig114+2017 look good. I'm going to PCR off the middle to check if the insides are go too. I used ca56 for ig114+2017 (binds to pbad promoter). I designed a primer for the middle of

Amy N. Kristofferson 13:24, 14 July 2010 (EDT)

To do:

• Redo cotransformation of Tera'a Payloads with Payload delivery devices in 1601K. DON'T FORGET MG!!!
• Run colony PCRs for KA transfer
• Run colony PCRs for SL/VB Assembly colony picks
  • Miniprep everything that wasn't co-transformed

Sequencing Submissions: the rest of my gel-purified assembly minipreps

iGEM51-56= each part w/ ca998 and g00101

Cotransformation Redo

Rescue started at 11;32am.

Sequencing Results for 2114+2316 (iGEM10_043): Great news! Successfully put Pcon in front of VBa

I submitted the part with ca998 and g00101, and the results clearly show that the Pcon is in front of the vacuole buster. Woo hoo!

KA transfer

Christoph Miniprepped everything he needed.

I miniprepped the remaining 1mL of 10 or bjh2294-PMLLKA (eluted with 25uL to keep normal MP concentration), ef1a-PMLLKA b, atub-PMLLKA b,c.

Parent Vector bleed-through results: All samples from 1,2,3,4,6,7,8,12,13,16 are good. The only samples with co-transformation were 15a and 9a,b,d.

Colony PCR- ca998+g00101

Tube	Lane	Quick Ref	part	expected length	good?
1--4	1,3,5,7	1	sbb09	235	all good
4--8	9,11,13,15	2	sbb07	1631	a,b,d
9--12	2,4,6,8	3	sbb06	441	all good
13--16	10,12,14,16	4	sbb04	1988	all good
17--20	17,19,21,23	6	sbb19	797	all good
21--24	25,27,29,31	7	sbb12	434	all good
25--28	18,20,22,24	8	sbb10	1226	all good
29--32	26,28,30,32	9	sbb42	291	all good
33--36	33,35,37,39	10	Bjh2294	2707	a,d
37-40	41,43,45,47	12	sbb04	1988	a,c
41-44	34,36,38,40	13	sbb19	797	a,d
45-48	42,44,46,48	15	sbb10	1226	all good
49-52	49,51,53,55	16	sbb42	291	a,b,c
53-68	57,59,61,63,50,52,54,56,58,60,62,64 (52,54,56-64)	5	Bjh1906	247	all good, except h
69-76	65,67,69,71, 73,75,77,79, 66, 68, 70,72 (65-73, 75,77,79)	11	Bjh2245	297	all good, except a, j
77-80	74,76,78,80	ef1a		1088	b
81-84	81,83,85,87	atub		1326	b,c,d

NOTE: lanes 49-96 refer to the middle gel. Just subtract 48 from the lanes in the table to see what number to refer to on the actual gel.

Colony PCR-th313+iGEM10

Note: the results for 5 are in the middle right gel shown above.

Tube	Lane	Quick Ref	Part	Expected length	Good?
1--12	89,91,93,95 and 82-96 even	5	Bjh1906	about 1650	all good, excect h
13-24	small gel: 1-12	11	Bjh2245	about 1651	b,c,e

SL/VB Assembly Colony Picks

Cotransformation results: 2h and 6a were co-transformed. Everything else had no growth on the triple antibiotics plate.

Column	Part	Plate Date	Lane	Expected lengths for ColPCR	Good lanes	Good wells	Miniprep
1	2114+2275	8-Jul	1-15odd	3622	possibly 11,13,15	a,b,c (I accidentally added the samples in the inverse order to the gel)	a,b
2	2114+2275	7-Jul	2-16even	3622
3	2271+2017	8-Jul	17-31 odd	2895	possibly 17, 19	a,b	a,b
4	2271+2017	6-Jul	18-32even	2895
5	ig114+2017	8-Jul	33-47 odd	3125	43	f	e,f
6	ig114+2017	6-Jul	34-48even	3125

Amy N. Kristofferson 18:16, 13 July 2010 (EDT)

Co-transform jh2348/2291-1601K with Tera's Payload

Added 100uL of H2O to 1uL of DNA from Tera's Ef1a and Atub payload constructs.
Added 10uL of H2O to 1uL of DNA from my minipreps of 2348/2291-1601K.

Added 1uL of each of those dilutions to 70uL of MG1655 cells.

Mg needs to be added for all steps of cloning the PDD's
1M MgSO4
10µL into rescue step (after heatshock)
400µL spread on plate (if +Spec, add 20µL and spread)
30µL into ea mL TB media (use TB for growing up for assay)

Started rescue at 4:06pm.

KA Vector transfer: ColPCR (parts and vector backbone check), inoculate, grow up

I picked colonies, inoculated and ran a colony pcr for all the parts we moved into the new, CORRECT KA vector.

Important notes:

• 5 and 11 were originally in AK vectors. 10 was also in an AK vector, but I gel purified it. Even so, I will run an analytical PCR of my minipreps of 10, and I ran an analytical Colony PCR with th313 and g00101 for my 5 and 11 colonies. I picked 12 colonies of 5 and 11.
• I also check for parent vector bleed-through by plating all the parts on spec, except for 9 (plated on triple antibiotics since it came from CK) and efla+atub (PCR products)
• All the inoculations are in the shaker.
• The colony PCRs are in the black PCR machines. They'll probably be done around 6pm.

SL/VB Assembly: Picking colonies from 7/6, 7/7, and 7/8 plates

Picked 8 colonies from Assembly attempt #2 (one pot) and assembly attempt #3 (gel purification) for 2114+2275, ig114+2017 and 2271+2017. Inoculated, checked for co-transformation, and set up ColPCR.

Amy N. Kristofferson 14:43, 12 July 2010 (EDT)

IMPORTANT UPDATE: TUBE MARKED PMLL6 (KA) in Eco/Bam Box is INCORRECT

Christoph just ran and analytical digest/gel that proved that the vector in the tube that was supposed to contain KA actually contained AK vector instead of KA. I need to Eco/Bam transfer the parts into a new KA vector. Also, this explains why the assembly with the Self-Lysis Device didn't work. We need to Eco/Bam transfer it into a KA vector too.

Digesting the new pMLL-KA backbone

Marianna gave us 10uL of her CMED7 in KA part. The dropout will be 324bp. To digest all of it, I multiplied the Analytical Mapping protocol by 2.5:

10uL of Water
10uL of CMED7-PMLLKA Miniprep
2.5uL NEB
1.25uL EcoRI
1.25uL of BamHI
Total: 25uL
Load 12.5uL into 2 wells. Gel Purify. Elute each with 12.5uL of H20.

Started digest at 4:30pm.

Ligation of ef1a and atub (bjh2342) eco/bam zymo'd digest

Ligated ef1a and atub eco/bam zymo'd digests (from my previous insertion into the supposed KA vector) with the new gel purified Eco/Bam digested KA vector. Started at 7:05pm. Transformed jtk030, MC1061 pir+ cells. In retrospect, I could have used Lefty.

Checking vector backbones of SL/VB Eco/Bam transfers

Set up a 2K55 PCR reaction with Taq MM, 1uL of 1/10 th313 oligo (in the middle of AmpR) and iGEM10 (rev oligo right before EcoR1 site) to verify the order on antibiotics on the backbone. This reaction PCR's off the AmpR gene to right before the EcoRI site. So if the vector is actually A-, the PCR product will contain a bit of AmpR and plasmid backbone and will be 865bp. If the plasmid is -A, the PCR product will contain a bit of AmpR, lots of plasmid backbone and the other antibiotic. In the case of CA, this amounts to 1808bp.

See Eco/Bam page in Excel notebook.

This data suggest that all the backbones that should be A- are indeed A- and that all the backbones that are -A are indeed -A. So the antibiotics shouldn't be the problem. For whatever reason, nothing turned up in the 2345 lane. I'll run the PCR again just to double check.

Lane	Part	Color	Expected length	Good?
1	Bjh2345	AC	865	no-empty lane
2	Bjh2271	AK	865	yes
3	Bjh1968	AC	865	yes
4	Bjh2316	AK	865	yes
5	Bjh2275	AK	865	yes
6	Bjh2114	CA	1808	yes
7	ig114	AK	865	yes

Lane Part Color Expected length Good? 1 Bjh2345a AC 865 no-empty lane 2 Bjh2345b AC 865 good 3 Bjh2345c AC 865 good

Threw out Bjh2345a. Must be a bad miniprep. I should probably map b and c. I guess I haven't yet...

Analytical digests of SL/VB Gel Purf. Assembly Minipreps

Eco/XhoI

Eco/BamHI

Tube	Part	Digest	Color	Expected Lengths (part, plasmid)	Good?	Second Digest	Expected lengths	Good?
1	2114+2316a	Eco/Xho	CK	4058, 1466	yes	started at 3:45
2	2114+2316c	Eco/Xho	CK	4058, 1467	yes
3	2114+2275	Eco/Xho	CK	4627, 1466		Eco/Bam (lane 1)	?	?
4	2271+2017	Eco/Xho	AC	3768, 1672		Eco/Bam (lane 2)	?	?
5	ig114+2017	Eco/Xho	AC	3998, 1672		Eco/Bam (lane 3)	?	?
6	iGEM16a+2294	Eco/Xho	CA	4644, 1466

Payload Delivery: Analytical Mapping of 2348 and 2291 in 1601K

Both parts look great! I'm going to give them to Jin.

Tube/Lane	part	Digest	Expected length	Good?
1	1601K-jh2348	Eco/Bam	4632, 2475	yes
2	1601K-jh2291	Eco/Bam	5927, 2475	yes

Eco/Bam Transfer from Bad KA to Good KA backbone

Lane	Part	short description	expected lengths	cut out
1	1601K-jh2348	payload	4632, 2475
2	1601K-jh2291	payload	5927, 2475
3	bjh2294	conor\'s self lysis	2868, 2516	lower
4	bjh1906	eco/bam plate: B11	46, vector (~2.8k)	zymo\'d (too small to purify)

Amy N. Kristofferson 21:13, 10 July 2010 (EDT)

Must do today:

• Miniprep good colony pcr/cotransformations from yesterday
• Pick colonies for Tahoura

If I have downtime, could start items in the above To Do list

From yesterday's colony picks, only the first colony picked from 16a H3 was cotransformed. No idea how that happened, since I gel purified the digests before ligating.

Amy N. Kristofferson 13:37, 9 July 2010 (EDT)

Colony PCR of SL/VB and 16a+SL Assembly

Results:

Very odd. Even though I gel purified the correct bands, it appears that only 2114+2316 worked correctly. I'm going to check to make sure that everything is in the correct vector, because I don't know what else could be causing a problem. Since I'm miniprepping a the couple correct products, might as well miniprep and map a few others to try and figure out what the problem may be.

Colony PCR	'	'	'	'	'
Tube	Combo	Expected length	Good?	Cotransformed?	Miniprepped
1	2114+2316	3053	yes		yes
2	\"
3	\"		yes		yes
4	\"
5	2114+2275	3622			yes
6	\"
7	\"
8	\"
9	2271+2017	2895			yes
10	\"
11	\"
12	\"
13	ig114+2017	3125			yes
14	\"
15	\"
16	\"
17	16a+H3	3566		yes
18	\"				yes
19	\"
20	16a+F3	3566
21

Animal Promoter: Analytical Mapping of atub and ef1a promoter in pMLLKA

Started an Eco/Bam digest of the 4 minipreps I made of each promoter (all of which had GREAT colony PCRs) at 10:35am. They will be ready to run on a gel at 11:05am.

Once it has been verified that these parts map well, I'll submit them for sequencing and then begin the next step of assembly.

Lane	Part	Digest	Expected Lengths	'
1	iGEM10_021 in PMLLKA	Eco/Bam	2868, 897	all good
2	Bjh2342 in PMLLKA	Eco/XhoI	2868, 1125	all good

Submitted ef1a-a and atub-b

Sequencing submission:

iGEM35	ef1a animal promoter (iGEM10_021 in PMLLKA)
iGEM36	atub animal promoter (jh2342 in pMLLKA)

Amy N. Kristofferson 14:36, 8 July 2010 (EDT)

Cotransformation check

None of the 2271+2017 or ig114+2017 colonies were cotransformed, but ALL of the iGEM10_018 (16a+SL) were cotransformed.

Colony PCR results for 2271+2017, ig114+2017 atub and ef1a promoter, iGEM10_16a

Can disregard the results for 16a+SL since all samples were cotransformed. 2017 combo inoculations are useless since nothing showed up on the colony pcr. Very strange, since they weren't co-transformed. GOOD NEWS: atub and ef1a look great.

Tube	Part	Lane	Expected length	Good
1--8	2271+2017	1-15odd	2895
9--16	ig114+2017	2-16even	2925
17--24	atub	17-31 odd	1326	19, 25, 29, 31
1a-8a	ef1a	18-32even	1088	all good
9a-24a	16a+SL	31-39	3566	tube 16a looks good (40th well, 8th sample), but they\'re all cotransformed.

Redo of SL/VB and 16a+SL digests with Gel Purification

To avoid co-tranformation and hopefully encourage the creation of the parts I want, I'm going to run a preparative gel, select the appropriate bands and gel purify.

Performed a Lefty (Bam/XhoI digest), Righty (Bgl/XhoI digest), and one-pot digest on all the parts in my SL/VB promoter as well as iGEM10_016a and Bjh2294 in PMLL-KA can be found in the Cheshire Chat plate in H3 and F3. I'll gel purify the Lefty digests of Bjh2345, Bjh2271, Bjh1968, Bjh2114, ig114, 16a and the Righty digests of Bjh2316, Bjh2275, and the two self-lysis devices. The other digests are just to make sure everything was methylated correctly and that the one-pot reaction works on these vectors.

Notes on protocols: Used Manual 2ab Assembly protocols for Lefty and Righty Mastermixes. Used my One-pot recipe, except with 0.4uL of each enzyme. Also, the DNA taken from the Cheshire Cat Plate was diluted by half b/c it was eluted with 100uL, so we had to adjust the amounts of NEB2 and enzyme used so that the recipe in the R/L digests turned out to be 1.6uL NEB2 and 1.5uL of each enzyme total, since our using 8uL of DNA instead of 4uL of DNA would otherwise have diluted the buffer and enzyme.

Digests started at 3:35pm.

From left to right: Lefty Digests, Righty Digests

Triple Digests

Results:

Lane	Part	vector	Purify	L Fragments (if no methylation for R\'s)	R Fragments (if no methylation, for L\'s)	One Pot fragments (if not methylated)	Proper methylation?
1	ig114a	pMLL7-AK	L-Upper	2925, 1196	2440, 1681	1681, 1244, 1196	Yes
2	Bjh2114	pMLL9-CA	L-Upper	1518, 1270	1476, 1312	1476, 1270, 42	Yes
3	Bjh2271	pMLL7-AK	L-Upper	2695, 1196	2210, 1681	1681, 1196, 3891	Yes
4	iGEM10_016a	pMLL5-CK	L-Upper	2333, 1196	2054, 1475		N/A (not methylated)
5	Bjh2316a	pMLL7-AK	R-Upper	4491, 1196	4006, 1681	2810, 1681, 1196	Yes
6	Bjh2275a	pMLL7-AK	R-Upper	5060, 1196	4575, 1681	3379, 1681, 1196	Yes
7	Bjh2294 F3	pMLL9-CA	R-Upper	4114, 1270	3777, 1607	2507, 1607, 1270	Yes
8	Bjh2294 H3	pMLL9-CA	R-Upper	4114, 1271	3777, 1608	2507, 1607, 1271	Yes

NOTE: lane 2 in the lefty gel is Bjh2294 H3, and everything else is pushed over one lane

Summary: Everything is methylated as it should be. I cut out all the right bands and purified them. Also, this shows that triple digestion isn't very efficient and cause a lot of parent vector bleedthrough (the top band of all the lanes). Also, note how dilute the SL DNA is... I'll still elute with the standard 8.5uL to concentrate the DNA, but this is still a potential problem.

Ligation and Transformation of SL/VB and 16a+SL purified digests

Started ligation at 6:45pm. Will transform into jtk030 (pir +, MC1061 cells). I forgot to digest 2017, so I used a righty digest that I had done previously. Started rescue at 8:10pm.

Amy N. Kristofferson 13:45, 7 July 2010 (EDT)

All the samples from 16a,b + SL were cotransformed, and since Jin mentioned that making a mastermix for the onepot reaction may result in there not being enough enzyme per reaction, I'm going to do it over again without making a mastermix AND using 0.5uL of enzyme instead of o.3uL of enzyme. This should cut back on parent vector bleedthrough as well.

There are a significant number of colonies on the plate for the 2271+2017 and ig114+2017 redo. I'll pick colonies/colony pcr/inoculate/check for cotransformation for 8 colonies from each. Also, since all the colony PCR's failed for my re-picking of 2114+2275, I'm going to redo the one-pot reaction for that as well.

The ligation of the atub promoter to the KA vector (Bjh2342-pMLL6-KA) was very successful, since I got a lot of colonies. I'll inoculate and run colony PCR (no need to check for co-transformation. Since my two colony picks of ef1a seemed to fail on the colony PCR yesterday, I'm going to pick a bunch more, inoculate, and run colony PCR again.

SL/VB Assembly: Redo of One Pot (with more enzyme) for 2114 combos

• One Pot (used 0.4uL of enzyme):
  • 2114+2275
  • 2114+2316
    • Will be ready for ligase at 2:00pm. Added ligase at 2:00pm. Will be ready for transformation into RIGHTY cells at 2:30pm. Rescue started at 3:30pm.

iGEM10_020 Assembly: redo of 16a+SL assembly, starting with new L/R digests

• Redo of L/R digest of 16a and 6+24
  • Put in incubator at 12:14pm. Ready for Zymo at 1:14pm. Zymo'd and added ligase at 2:00pm. Will be ready for transformation into RIGHTY cells at 2:30pm. Rescue started at 3:30pm.

Possibly successful assemblies (Animal Promoter, SL/VB and iGEM20

• Picked colonies (8 each)/inoculate/cotransformation check
  • 2271+2017
  • ig114+2017
  • 16a+SL (pick at least 12 NEW colonies)
  • atub and ef1a (no cotransformation check)

Miniprep of Payload Delivery Vectors

Miniprepped Payload Delivery stuff (in shaker w/ Mg LB)

Sequencing Results for iGEM10_017b, 2114+2316b,Bjh2345 PMLL4

Sequencing results:

• iGEM10_017b: the first 990bp or so of the 1546bp part are perfect, so I think it's safe to say that this miniprep is good to use for future assembly.
• 2114+2316b: this proved to have the Pcon, but no vacuole buster in the part. At first I thought this was odd, since the part miniprepped well, but it makes sense now. The part had a bad colony PCR since only the 43bp pCon was in the vector. The part had what looked like a good miniprep map since the vector was the same size as the part, and I when I saw a band, I assumed it was the part AND the vector. My mistake.
• Bjh2345 PMLL4: bad read. Do again, but make sure concentration is accurate?

Amy N. Kristofferson 13:49, 6 July 2010 (EDT)

Colony PCR results for all colonies picked today

Note: they ALL look horrible. Bands in the 16a lanes are probably from cotransformation with 16a vector, since that part is about 850bp.

Lane	Part	Expected length
1	2114+2275 repick	3622
2	\"
3	\"
4	\"
5	\"
6	\"
7	\"
8	\"
9	16a+SL	3566
10	\"
11	\"
12	\"
13	16b+SL	3566
14	\"
15	\"
16	\"
17	ef1a	1088
18	\"

Transposase Assembly: Colony PCR of iGEM10_016a,b with Self Lysis Device

Picked 4 colonies of each. Ran Colony PCR. Inoculated. Plated to check for co-transformation.

Animal Promoter: Colony PCR of Ef1a

Picked 2 colonies. Ran Colony PCR. Inoculated.

Conor Eco/Bam digested and zymo'd the atub promoter (Bjh2842). Started ligation with PMLL6-KA vector at 4:45pm. Transformed into jtk049 cells. Started rescue at 5:55pm.

Payload Delivery

Since the payload delivery device contains the Self Lysis device under a promoter that's induced in low levels of Mg, I need to grow up my colonies in LB containing 30mM Mg to prevent Lysis. In the future, I should also rescue/plate on Mg. I selected clear colonies.

Tomorrow, I will miniprep the inoculations and map the minipreps next to the parent vector containing the part, so I can make sure the whole part is in the new vector. (Jin said recombination events can occur which could shorten the part)

SL/VB Promoter Assembly

Redo one pot reaction with 2271+2071 and ig114+2071

Conor set up two one pot reactions and put them in the thermocycler under my ONEPOT program. Added ligase at 4:45pm. Will transform into LEFTY cells. Started rescue at 5:55pm.

Picked more colonies for 2114+2275, since miniprep mapping failed

Pick 8 more colonies from 2114+2275. Ran colony PCR, inoculate, check for cotransformation.

Summary of Results so far

Part	Transformation	Miniprep Map	Colony PCR for that Miniprep	To do
2114+2316	good	good	bad	Seq
2114+2275	good	bad	bad	repick colonies
2271+2071	bad	N/A	N/A	redo one-pot
ig114+2071	ok	bad	good	redo one-pot
2345-pMLL4	good	good	bad	Seq

For whatever reason, my Colony PCR results and miniprep results don't seem to ever correspond.

Amy N. Kristofferson 19:16, 5 July 2010 (EDT)

SL/VB Promoter Assembly: Mapping the minipreps produced from Round 1 and 2345 Redo

Started digestion at 5:18pm. Will be ready for the gel at 5:48pm.

Tube	Part	Methylated?	Digest	Color	Expected Lengths (part, plasmid)	Good?	Sequence	Next step
1	2114+2316a	R	Eco/Xho	CK	4058, 1466	good	yes
2	2114+2316b					possibly good	yes
3	2114+2275b	R	Eco/Xho	CK	4627, 1466	BAD		Pick more colonies
4	2114+2275c					BAD
5	ig114+2017	L	Eco/Bam	AC	2925, 2745	BAD		redo
6	2345 in pMLL4a	L	Eco/Bam	AC	2680, 2745	good	yes
7	2345 in pMLL4b					good
8	2345 in pMLL4c					good

Transposase Assembly: Ligation of iGEM10_016a,b Lefty digest with 6+24 Righty digest

Started ligation at 4:05pm. Ligated unmethylated 6+24 digested with BglII+XhoI with both unmethylated iGEM10_016a+b, which had been digested with BamHI+XhoI. Tubes are labelled 16a+SL and 16b+SL (SL meaning Self Lysis, which is part 6).

After ligation is over, I will transform into RIGHTY cells. Started rescue at 5:05pm. Plated on CA.

Animal Promoter: Ligate Ef1a Eco/Bam digest of PCR product with PMLL6-KA

Started ligation at 4:05pm. Ligated Ef1a Eco/Bam digest of PCR product with pre-digested PMLL6-KA.

After ligation is over, I will transform into LEFTY cells. Started rescue at 5:05pm. Plated on KA.

Payload: Ligation of Bjh2348 and 2291 with Pjh1601K

Started ligation at 4:05pm. Ligated Eco/Bam digests of Bjh2291 and 2348 with my Eco/Bam digested Pjh1601K plasmid.

After ligation is over, I will transform into jtk049 cells, because they don't need to be methylated. Started rescue at 5:05pm. Plated on K.

Amy N. Kristofferson 13:00, 2 July 2010 (EDT)

Transposase Assay Take 2: Testing time for Transposase expression and time for transposase activity

1. Each inoculation contains about 4mL of cells. Move 1000uL of cells into four flasks.
   1. Each flask contains 900uL of cells.
2. Add 10uL of arabinose for each mL of cells at t=o, 1 hour, 2 hours, 3 hours.
   1. Since arabinose is 100x, will add 9uL of arabinose to each flask
   2. Added first 9uL at 10:49am.
   3. Added second 9uL at 11:49am
3. After four hours have passed since you first added arabinose, add 1uL of atc per mL of cells. Allow 1 hour for lysis.
   1. Will add 0.9uL of atc, since 900uL of cells are being used and the concentration of the atc is 1000x.
4. After that one hour, add 5uL of DNA to each tube.
5. Remove a 200uL sample of lysate after 30min, 1 hour, 1.5hour, 2hours.
6. Immediately after removal, spin down the lysate and zymo the supernatant.
7. Transform lefty pir+ cells with the lysate. Plate on Amp and Gen.

Unfortunate update: Sequencing proved that iGEM10_020b is a dud. Gibson didn't work because one of our junctions contained palidromic terminal repeats, so the oligos designed based off those junctions didn't have a unique binding site. Oh well. We'll proceed with manual 2ab assembly.

iGEM10_20 2ab Assembly

Mapping of iGEM10_016 minipreps a and b

An Eco/Bam digest of iGEM10_16 in PMLL5-CK should result in bands at 2662 and 867.

Note: iGEM10_017 has already been mapping. (see below) Results were slightly confusing, so I'll submit it for sequencing...? iGEM10_001 was also mapped, and looks good. 1a-c are all good options.

Started digestion at 4:55pm.

Both have bands at the appropriate places, but 16b has a lot more parent vector remaining, for whatever reason, so 16a is probably the best choice.

Assembly of iGEM10_018

Lefty Digest of iGEM10_16

Righty Digest of Self Lysis Device (6+24 taken from well G10 of ROMEO plate) Started at 4:42pm. Zymo'd and stored in my working box.

SL/VB Promoter Manual Assembly: Analysis of One-Pot reaction

2114+2316 and 2114+2275 both successfully transformed righty pir+ cells. The plates are covered in colonies. 2271+2017 and ig114+2071 weren't as successful. The former had no colonies, while the latter had 11, which is surprising, because I used barely any DNA.

I will pick four colonies from each, colony pcr, inoculate, and check for cotransformation.

Cotransformation results:

The ones with letters in the corners were chosen for minipreps.

Tube	Combo	Expected length	Good?	co-transformed?	miniprep?
1	2114+2316	3053	yes	no	yes
2	\"			no	yes
3	\"
4	\"			no
5	ig114+2017	3125	yes	no	yes
6	\"		yes
7	\"		yes
8	\"
9	2345 in pMLL4	2880		no	yes
10	\"			no	yes
11	\"			no	yes
12	\"			no
13	2114+2275	3622
14	\"			no	yes
15	\"			no	yes
16	\"

Moving into jh1601K

Performed an Eco/Bam Digest of jh1601K. Put in incubator at 11:09am. Trashed that digest. Realized I need to do a gel purification, so I'll do the digest again with 4uL of plasmid.

Started digest again at 4:54pm.

2345 Redo from 24+PMLL4 digests

The plate is covered in colonies. I will pick 8 colonies, inoculate, and check for cotransformation.

Amy N. Kristofferson 13:40, 1 July 2010 (EDT)

Eco/Bam transfer 2348 and 2291 into 1601K

Started Digest at 12:19pm.

Zymo'd and stored in my working box.

2345 Redo: Ligating Tim's ebid 24 and EB pMLL4-AC

Since my Colony PCR turned out so horribly yesterday, and 2345 is the Self Lysis Device we've been using for all our construction, I'm just going to use Tim's digests and ligate them together.

Started ligation at 11:48am.

I transformed the plasmid into lefty pir+ cells. Started rescue around 12:30pm. Plated on AC at 2:13pm.

One Pot Assembly of Jin's Parts

I'm going to redo the construction of the following with a one pot reaction, since they're all methylated.

Lefty Digest	Righty Digest	Transform into	Plate on	Short Description	'
2114	2316	R	CK	pCon.VacuoleBusterA
2114	2275	R	CK	pCon.VacuoleBusterB
2271	2017	L	AC	Pbad.SelfLysisT
ig114	2017	L	AC	Ptet.SelfLysisT

Started digest at 11:10am, for all but ig114+2017. I added the speck of 2017 (~.2uL) left at around 11:36am. Started the 20 min heat kill for all but ig114+2017 at 12:30pm. Accidently set the heat kill program for 20 sec instead of minutes and added ligase. Hopefully that won't hurt anything. Put all four tubes in the PCR machine to heat kill at 12:53pm. Added ligase at 1:41pm. Started rescue at 2:42pm.

These numbers have been based on Qiagen minipreps of Jin's pBjh1601 assembly vectors:

• Set up the digestion:

1uL lefty plasmid(methylated)
1uL righty plasmid (methylated)
1uL NEB2+ATP
0.3 uL XhoI
0.3 uL BglII
0.3 uL BamHI
6.1uL Water

• Digest for 1 hour at 37C
• Heat kill at 65 for 20 min
• Add 0.3uL T4 Ligase
  • Ligate at room temperature for 30 minutes
• Put on ice for transformation
• Transform, rescue, plate on dual antibiotic plates

Notes:

• This requires the use of BglII and BamHI methylating strains!
• 10X "NEB2+ATP" is a homemade buffer present as aliquots in 500uL

tubes labeled with a red slash

• You can make up a mastermix of the enzymes/buffer/water and

distribute the plasmids into it

• The leftmost thermocyler in 327 has a program JCA/1123 that runs the

1hr at 37 and heat kill steps of the procedure

Eco/Bam digest of EF1a PCR product

Started digest at 11:55am. Zymo'd the product.

I need to figure out what assembly vector to put it in and then map and sequence the part to make sure it's correct.