Berk2010-Conor

June Entries

To Do:

• Choano Assays
• next stage of 2AB assembly for parts 007 and 013

Conor McClune 15:47, 23 July 2010 (EDT)

Restriction mapping of iGEM10_006 Eco/Bam

• digested all 5 minipreps with Eco/Bam
• 4uL DNA, 4uL H20, 1uL each enzyme, 1uL NEB2
• incubated 11:15-12:12

Assembly of iGEM10_012

Digestion
Digested iGEM10_010 with lefty MM and iGEM10_011 with righty MM

• incubated 11:25-12:25

2:00-

• expected sizes:
  • 010:
    • 2912, 1064
  • 011:
    • 4834, 1475

Gel Purification

'Ligation
I did a standard ligation recipe and let it sit from 4:00-4:30

Repicking Colonies of iGEM10_006 Eco/Bam Transfer

I may have destroyed my DNA samples by vortexing during the wrong stage of miniprepping, so I am going to grow them up again. There are only 5 colonies, and we know from the last colony PCR that they are all on the correct vector. This time I shall run a Col PCR that will check the part.

• Repicking all 5 colonies
• running col PCR with oligos: ca998 and igemTen004

Gel Analysis of iGEM10_006 Eco/Bam digest

Expected size: 5925

Conor McClune 17:24, 22 July 2010 (EDT)

Picking Colonies/ Colony PCR for iGEM10_007

• Picking 24 colonies
• colony PCR's:
  • ca56 + igemTen004
    • checks for iGEM10_001 and sbb04 (left side of iGEM10_007)
    • expected size: 2045

Gel Results

• all lanes are empty

Conor McClune 13:52, 21 July 2010 (EDT)

NH4 Toxicity Assay

Setup (completed 9:15am)

• made 4 aliquots of 3mL of healthy MbFb (2 days after 1:15 split)
• placed these in the following conditions:
  • 25*C
  • 37*C
  • 37*C with 10mM NH4
  • 37*C with 1mM NH4

1 Hour Count (10:15am)

• the PI stain seems to be dead, because I can see no fluorescence, including background. Thus I will only take pictures and qualitative observations
• increased swimming in all three 37*C samples, otherwise, no difference between samples

4 Hour Observations (1:15 pm)

• choanos are still much more active at 37*C than at 25*C

12th Hour (9:15pm)

Choanos are still active in all wells. I took a hemocytometer count of the cells in each wells. However, I did not dilute the first two cultures (which were denser than I expected) and counting took longer than 30 minutes (the usual time before formaldehyde begins causing choanos to lyse). Thus, the counts for the last two cultures may be inaccurate.

Choano Counting Procedure:

• scrape choanos
• remove 200uL of each of the 4 cultures to 1.5mL tubes
• add 1uL formaldehyde to each tube, shake
• pipette 10uL of each tube into hemocytometer
• wait 10 minutes for choanos to sink
• count 4 quadrants and average:

Note: due to time crunch, I diluted the two NH4 samples 1:10 in order to speed up the process

'	Dilution	1	2	3	4	Average	Cell density (cells/mL)
25*C control	n/a	242	309	249	270	267.5	2675000
37*C control	n/a	284	321	290	274	292.25	2922500
37*C 10mM NH4	1 :10	27	31	29	6	23.25	2325000
37*C 1mM NH4	1 :10	17	30	17	24	22	2200000

At 9:30, I had scraped the cells to do the above counts, so by 10:30 the percentage of choanos attached the the bottom had peaked. I aspirated all the media from each of the 4 cultures and replaced it with 3mL choano media.

25th Hour

I removed 100uL of each culture, diluted it 1:4 and counted it on the hemocytometer. This should be a more accurate count than the one at 12 hours.:

'	Dilution	1	2	3	4	Average	Cell density (cells/mL)
25*C control	1 :4	31	29	31	25	29	1160000
37*C control	1 :4	13	16	16	16	15.25	610000
37*C 10mM NH4	1 :4	14	11	10	7	10.5	420000
37*C 1mM NH4	1 :4	16	15	12	11	13.5	540000

Results: The choanos clearly recovered from the NH4. At this point it looks like the temperature difference is more detrimental to growth than NH4.

Picked colonies for iGEM10_011

Picked 8 colonies and ran a Col PCR with standard Col PCR MM (caa998 + G00101)

PCR Gel Results:
Expected size: 3700bp

• lanes 3-8 look perfect
• Colonies 1-2 are cotransformed.

To miniprep: iGEM10_011 #3,4

Miniprepped iGEM10_006 #1-5

Note: Vortexed briefly after adding P2, which may cause some contamination with genomic DNA.

Assembly of iGEM10_007

Digestion

• iGEM10_001 a - did standard Xho/Bam digestion
• iGEM10_006 #1-5:
  • digested all 5 minipreps; this digestion will serve as both analytical and practical.
  • made a master mix: 20uL H20, 5uL NEB2, 5uL each of Bgl and Xho
  • mixed 7uL of mastermix with 4uL of each miniprep

I decided to gel purify the gels, since I was running a gel. Expected sizes:

• iGEM10_006: 6994, 1608
• iGEM10_001: 2810, 1196

Conor McClune 20:08, 20 July 2010 (EDT)

Reassembly of iGEM10_011

iGEM10_011 once again did not grow, so I suspect my digestion of iGEM10_008 may be bad and should be redone.

Digestion
Did a standard Xho/Bam digestion of iGEM10_008d and gel purified the larger part (2.5kb)

Ligation
Ligated my gel purified fragment with Amy's gel purified Xho/Bgl digestion of Bjh2294.

Transformation
Cell mixture: 100uL jtk030 cells, 30uL kcm, 50uL h20. I added 90uL of this mixture to my ligation mixture, incubated for 1hr and plated the entire solution on a CA plate.

Picking Colonies for iGEM10_006 Eco/Bam Transfer into KA

Only five colonies grew, so I picked all 5. I swiped each colony on an AKC plate to check for cotransformation, and dipped it in a colony PCR mix containing the oligos igemTen10 and TH313.

PCR Gel Results
Expected size: 1808
Incorrect Size: 800

• all five parts appear to be in the correct vector.
• None of the colonies were cotransformed

Miniprep of iGEM

I miniprepped iGEM10_004 #8 and iGEM10_010 #1-3.
note:I accidentally used Zymo tubes (which decreases yield), so I only eluted with 25uL H20

Restriction Mapping of iGEM10_004,iGEM10_005, iGEM10_010

Yesterday's Minipreps

Lane	Part	Colony #	Vector	Enzymes	Expected Sizes	Acceptable?
1	iGEM10_004	8	pMLL9-CA	Eco/Bam	3580, 2736	probably
2	igEM10_005	1	pMLL8-KC	Eco/Bam	2662, 2163	yes
3	igEM10_005	5	pMLL8-KC	Eco/Bam	2662, 2163	yes
4	igEM10_005	6	pMLL8-KC	Eco/Bam	2662, 2163	yes
5	igEM10_005	8	pMLL8-KC	Eco/Bam	2662, 2163	yes

Today's Minipreps

Lane	Part	Colony #	Vector	Enzymes	Expected Sizes	Acceptable?
1	iGEM10_004	8	pMLL9-CA	Eco/Bam	3580, 2736	probably
2	igEM10_010	1	pMLL8-KC	Eco/Bam	2662, 1314	yes
3	igEM10_010	2	pMLL8-KC	Eco/Bam	2662, 1314	yes
4	igEM10_010	3	pMLL8-KC	Eco/Bam	2662, 1314	yes

Conor McClune 15:37, 19 July 2010 (EDT)

Sequence Confirmation of iGEM10_006 #3

I sequenced iGEM10_006 #3 with the reverse oligo igemTen008. This part does not quite reach the junction I was trying to make. I will try sequencing it again using igemTen003.

Eco/Bam Transfer of iGEM10_006 #3

Digestion
4uL H20, 4uL DNA, 1uL Eco, 1uL Bam, 1uL NEB2. Digested from 11:05-12:10
I also needed to digest more pMLL6-KA, since our stock is empty. to do this, I mixed 10uL miniprepped DNA (containing pMLLKA) with 10uL H20, 2.5uL NEB2, 1.25uL Eco, 1.25 Bam. Digested from 1:49-2:23

Gel Purification
Expected bands:

• iGEM10_006
  • desired - 5700
  • undesired - 2800
• pMLL6-KA
  • desired - 2877
  • undesired - 324

Zymo purified the desired products. Eluted iGEM10_006 with 8.5 uL water, and pMLL6-KA with 20uL water

Ligation
Standard ligation procedure and recipe.

Assembly of iGEM10_011

No colonies grew up for iGEM10_011, so I have to re-assemble it. I already have a gel purified

Picking Colonies for iGEM10_004 and iGEM10_010

• No colonies grew up for iGEM10_011.
• 8 Colonies picked from each plate.
• iGEM10_010 colonies dipped in a colony PCR MM with oligos ca998 and G00101
• iGEM10_004 colonies dipped in two Col PCR MM:
  • ca998 and G00101
  • igemTen10 and TH313

PCR Gel Results

• cotransformation plate reveals that iGEM10_004 colonies #1 and #4 are cotransformed

Lanes	Part	Vector	Fwd Oligo	Rev Oligo	Expected Size	Acceptable Lanes
1 to 8	iGEM10_004	pMLL9-CA	ca998	G00101	3771	8 (possibly)
9 to 16	iGEM10_004	pMLL9-CA	igemTen10	TH313	1808	9,12,16
17 to 24	iGEM10_010	pMLL8-KC	ca998	G00101	1505	17-19,21-24

to miniprep: iGEM10_004 #8, iGEM10_010 #1-3

Conor McClune 16:00, 16 July 2010 (EDT)

Assembly of iGEM10_010-11

Digestion:
iGEM10_009 and iGEM10_008 are already digested and gel purified

• used methylated 2:1 diluted minipreps of Bjh2294 and sbb10
• recipe:8uL DNA, 4uL water, 1uL NEB2, 1uL each enzyme
• digested sbb10 with Xho/Bam, digested Bjh2294 with Xho/Bgl
• digested from 12:50-1:40
  

Ligation

• Recipe: 6.5uL H20, 1uL each DNA, 1uL T4 Buffer, 1uL Ligase
• I ligated the following combinations using the above digestions and stored digestions from my freezer box:

Part 1	Part 2	Product
sbb10	iGEM10_009	iGEM10_010
iGEM10_008	Bjh2294	iGEM10_011
iGEM10_004	pMLL9-CA	iGEM10_004

Transformation

• started with 100ul comp jtk030 cells
• added 100ul H20 and 40uL KCN
• added 70uL of cell mixture to each ligation
• conducted standard heat shock transformation with 1hr incubation
• plated iGEM10_010 on CK and iGEM10_011/iGEM10_004 on CA

Analytical PCR of iGEM10_006 #3

Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA

Well	Part	Fwd Oligo	Rev Oligo	Expected Size	Acceptable?
1	iGEM10_006	ca998	igemTen004	1859	yes
2	iGEM10_006	ca998	igemTen006	2225	yes
3	iGEM10_006	ca998	igemTen008	3289	yes

Conor McClune 15:43, 15 July 2010 (EDT)

Picking Colonies/Colony PCR for Eco/Bam transfers (sbb18, iGEM10_004-5)

• picked 8 colonies of sbb18, iGEM10_004, iGEM10_005
• Two PCRs per colony:
  • sbb18:
    • ca998+G00101
    • TH313+igemTen10
  • iGEM10_004:
    • ca998+G00101
    • TH313+igemTen10
  • iGEM10_005:
    • ca998+G00101
    • MLL2F+igemTen10
• TH313 is a fwd primer out of AmpR and MLL2F is a fwd primer out of CamR
• I swiped each colony on an ACK plate to check for cotransformation

Gel Results

Lanes	Part	Vector	Checking	Fwd Oligo	Rev Oligo	Expected Size	Acceptable Lanes
1 to 4	sbb18	pMLL6-KA	part	ca998	G00101	599	1,2,3,4
5 to 8	sbb18	pMLL6-KA	vector	TH313	igemTen10	1939	1,2,3,4
9 to 16	iGEM10_004	pMLL9-CA	part	ca998	G00101	3771	none
17 to 24	iGEM10_004	pMLL9-CA	vector	TH313	igemTen10	1939	19,20,21,24
25 to 32	iGEM10_005	pMLL8-KC	part	ca998	G00101	2354	25,29,30,32
33 to 40	iGEM10_005	pMLL8-KC	vector	MLL2F	igemTen10	1808	33,35,37,38,40

Miniprep/Mapping iGEM10_006

• The colonies I picked yesterday were all on the wrong plasmid (AK not KA) and the internal plasmids did not seem to bind, but I will miniprep and digest, just in case the correct part is there within the wrong plasmid. The internal plasmids I used were long (designed for Gibson) and can be finicky.

Digestion

• standard Eco/Bam digestion (4uL H20, 1uL NEB2, 4uL DNA, 1uL each enzyme)
• digested all 5 minipreps

Gel Results<br?
Expected Sizes:5734, 2868

• ladder warping makes it difficult to be precise, but if any lanes are correct, it is lane 3

Conor McClune 14:33, 14 July 2010 (EDT)

Assembly of iGEM10_016

Picking Colonies and Colony PCR

• picking 8 colonies
• swiping on cotransformation CKA plate
• dipping each colony in two col PCR mixes:
  • igemTen003+igemTen008
    • checks the presence of both iGEM10_004 and iGEM10_005
    • expected size:1461
  • TH313+igemTen010
    • checks order of Amp and Kan resistance genes in backbone
    • desired (KA) size: 1939
    • undesired (AK) size: 874

Gel Results:

• lanes 1-5: colonies 1-5 with igemTen003+igemTen008
  • checks the presence of both iGEM10_004 and iGEM10_005
  • expected size:1461
  • no bands visible in any lane
• lanes 6-10: colonies 1-5 with TH313+igemTen010
  • checks order of Amp and Kan resistance genes in backbone
  • desired (KA) size: 1939
  • undesired (AK) size: 874
  • all colonies have undesired antibiotic configuration (KA)
  • no colonies were cotransformed

Eco/Bam Transfer sbb18 into correct KA part

Digestion: Standard Eco/Bam digestion of sbb18 from Romeo, incubated for 30 minutes.

Gel Purification:
sbb18:

• desired fragment-408
• undesired fragment-2868

iGEM10_004:

• desired fragment-3580
• undesired fragment-2868

iGEM10_005:

• desired fragment-2163
• undesired fragment-2868

I cut out the desired bands, dissolved them and zymo purified them.

Ligation:

• master mix: 24uL H20, 4uL T4 buffer w/ATP, 4uL Ligase
• 8uL master mix added to 1uL of vector and 1uL part:
  • sbb18 with pMLL6
  • iGEM10_004 with pMLL9
  • iGEM10_005 with pMLL8

Transformation:

• 100ul Lefty pir+ mc1065
  • added 30uL KCM to cells
  • add 70uL of cells/KCM to ligation mixture of sbb18
• 100uL jtk030 cells
  • added 50uL H20 and 40uL KCM
  • added 70uL of cells to ligation mixtures for iGEM10_004 and iGEM10_005

• heat shocked cells for 90 seconds
• added 100uL 2YT to each of the 3 cultures
• rescued for 45 minutes
• plated:
  • sbb18 on KA
  • iGEM10_004 on CA
  • iGEM10_005 on CK

Conor McClune 15:45, 13 July 2010 (EDT)

Assembly of iGEM10_006

Digestion

• iGEM10_005 #3: 4uL DNA, 4uL H2O, 1uL NEB2, 1uL Xhol, 1uL Bam
• iGEM10_004 #4: 4uL DNA, 4uL H2O, 1uL NEB2, 1uL Xhol, 1uL Bgl
• incubated from 11:19-12:25

Gel Purification

• iGEM10_004 #4:
  • desired fragment:4841
  • other fragment:1475
• iGEM10_005 #3:
  • desired fragment:3629
  • other fragment:1196

I gel purified the desired fragments and zymo purified them. I accidentally used double the water (17uL), so my zymo is a little dilute and I had to modify the ligation procedure

Ligation

• 2uL digested iGEM10_004 #4
• 2uL digested iGEM10_005 #3
• 4uL H20
• 1uL T4 ligase buffer with ATP
• 1uL T4 Ligase

Transformation

• 20uL KCM added to 100uL JTK030 comp cells
• all of ligation mixture added to these cells
• sat on ice for 10 minutes
• heat shocked at 42 for 90 seconds
• rescued at 37 on shaker form 2:56-4:02
• plated entire solution on KA plate

Conor McClune 14:14, 12 July 2010 (EDT)

Eco/Bam Transfering Basic parts to correct KA vector

• used Tim's digested parts and Amy's new digest pMLL6-KA
• I eco/bam digested sbb42 for 1hr and Bjh2245 for 30 min
• created ligase MM:
  • 130uL H20
  • 20uL T4 ligase buffer
  • 20uL Eco/Bam digested pMLL6-KA
• added 8.5uL MM to 1uL of each of the following Eco/Bam digested parts:

Well	Ebid	Part	Strain
1	2	sbb09	R
2	4	sbb07	R
3	5	sbb06	R
4	7	sbb04	R
5	11	Bjh1906	R
6	12	sbb19	R
7	19	sbb12	R
8	22	sbb10	R
9	23	sbb42	R
10	24	Bjh2294	R
11	na	Bjh2245	R
12	7	sbb04	L
13	12	sbb19	L
15	22	sbb10	L
16	23	sbb42	L

• Transformed into the appropriate righty or lefty mc1061 pir+ strain

Restriction Mapping

• digestion master mixes- 4 H2O: 1 NEB2 : 0.5 Enzyme1 : 0.5 Enzyme1

Well	Part	Vector	Enzymes	Expected Sizes	Acceptable?
1	sbb10 #1	pMLL6-KA	Eco/Bam	2868, 1035	yes
2	sbb10 #2	pMLL6-KA	Eco/Bam	2868, 1035	yes
3	sbb10 #3	pMLL6-KA	Eco/Bam	2868, 1035	yes
4	sbb10 #4	pMLL6-KA	Eco/Bam	2868, 1035	yes
5	iGEM10_004 #1	pMLL8-KC	Eco/Bam	2662, 2163	failed digest (righty)
6	iGEM10_004 #2	pMLL8-KC	Eco/Bam	2662, 2163	failed digest (righty)
7	iGEM10_004 #4	pMLL8-KC	Eco/Bam	2662, 2163	failed digest (righty)
8	iGEM10_005 #6	pMLL8-KC	Eco/Bam	2662, 2163	failed digest (righty)
9	sbb04 #1	pMLL6-KA	Eco/Bam	2868, 1797	yes
10	sbb04 #2	pMLL6-KA	Eco/Bam	2868, 1797	yes
11	Bjh2294 #1	pMLL6-KA	Eco/Bam	2868, 2516	probably
12	Bjh2294 #2	pMLL6-KA	Eco/Bam	2868, 2516	probably
13	iGEM10_005 #2	pMLL9-CA	Eco/Xho	3227, 1598	yes
14	iGEM10_005 #3	pMLL9-CA	Eco/Xho	3227, 1598	yes
15	iGEM10_005 #4	pMLL9-CA	Eco/Xho	3227, 1598	yes
16	iGEM10_005 #8	pMLL9-CA	Eco/Xho	3227, 1598	yes

iGEM10_004 failed to digest because Bam does not digest righty-methylated parts. I am redoing it with an Eco/Xho digest:

Well	Part	Vector	Enzymes	Expected Sizes	Acceptable?
1	iGEM10_004 #1	pMLL8-KC	Eco/Xho	4850, 1466	too faint to see
2	iGEM10_004 #2	pMLL8-KC	Eco/Xho	4850, 1466	too faint to see
3	iGEM10_004 #4	pMLL8-KC	Eco/Xho	4850, 1466	looks good
4	iGEM10_005 #6	pMLL8-KC	Eco/Xho	4850, 1466	looks good

Eco/Bgl/Bam assembly of iGEM10_006, iGEM10_010, iGEM10_011

Digestion

• digestion master mixes- 4 H2O: 1 NEB2 : 0.5 Enzyme1 : 0.5 Enzyme1

Part	Vector	Colony # or letter	Enzymes	Desired Frag	Other Fragment
iGEM10_005	pMLL8-KC	3	Eco/Bam	2163	2662
sbb10	pMLL6-KA	1	Eco/Bam	1035	2868
iGEM10_008	pMLL5-CK	d	Eco/Bam	1066	2662
iGEM10_004	pMLL9-CA	2	Eco/Bgl	6307	9
iGEM10_009	pMLL4-AC	b	Eco/Bgl	3015	9
Bjh2294	pMLL6-KA	1	Eco/Bgl	5375	9

• I gel purified all the desired fragments
• note, there was no clearly visible band at 6307 in lane 4, but I cut out a larger area. The above restriction digests of iGEM10_004 suggest that it may have been better to use colony #3 or 4, not #2, as I did.

Conor McClune 22:25, 11 July 2010 (EDT)

Col PCR results of picking iGEM10_004, iGEM10_005, sbb04 and Bjh2294

• lanes 1-8 : iGEM10_004
  • expected size:3771
  • wrong size, most lanes seem to match the size of iGEM10_005 instead
• lanes 9-16 : iGEM10_005
  • expected size:2354
  • none are correct size, though lanes 1,2,4,5,6 match the size of iGEM10_004
• lanes 17-20 : sbb04
  • expected size:1988
  • all 4 lanes look good
• lanes 21-24 : Bjh2294
  • expected size:2707
  • all 4 lanes look good

Note: lanes 17-24 and final ladder are somewhat warped due to the plastic object I used to hold the gel

I must have switched the plates for iGEM10_005 and iGEM10_004 when I was picking, so I have the correct parts under the wrong labels. From this point one I will switch the iGEM10_005 and iGEM10_004 back.

Conor McClune 20:15, 10 July 2010 (EDT)

Replenish stcoks of sbb10

• miniprepped all 4 colonies I grew up (though colony #1 looks best)

Replenish stocks of sbb04 and Bjh2294 (Self Lysis Device)

• picked 4 colonies of each and transferred to KA LB

Assembly of iGEM10_004, iGEM10_005 and iGEM10_011

• no colonies grew for iGEM10_011, but a few grew up on the plates for iGEM10_004 and iGEM10_005

Conor McClune 15:02, 9 July 2010 (EDT)

Retransform Bjh2294 (self lysis device) and sbb04 for stock replentishment

• used Assembly teams methylated, 1:2 diluted stocks
• transformed 4uL of of each miniprepped DNA into 100uL jtk030 comp cells (w/ 20uL KCM).
• rescued for 1hr at 37*C
• plated entire solutions onto KA plates

Replenish stocks of sbb10

• picked 4 colonies from yesterdays transformation and placed in 3uL KA LB
• set up a Col PCR with ca998 and G00101

Col PCR results:

• expected size:1226

Results:
Colony one looks great, other colonies show faint lines.

Assembly of iGEM10_004, iGEM10_005, iGEM10_011

Digestion

• made lefty and righty digestion MM
• digested the following parts:

Well	Part	Vector	MM	Desired Fragment	Other Fragment
1	sbb04	pMLL6-KA	L	3395	1270
2	iGEM10_003b	pMLL5-CK	L	2539	1064
3	iGEM10_008d	pMLL5-CK	L	2532	1196
4	iGEM10_002d	pMLL4-AC	R	1430	1681
5	iGEM10_009b	pMLL4-AC	R	1343	1681

• I gel purified the desired bands
• Amy already had gel purified a lefty (xho/bam) digest of Bjh2294

Ligation

• I did standard ligations to make the following parts:
  • sbb04 + iGEM10_002 = iGEM10_004
  • iGEM10_003 + Bjh2294 = iGEM10_005
  • iGEM10_008 + Bjh2294 = iGEM10_011

Transformation

• I transformed iGEM10_004 and iGEM10_011 into righty pir+ mc1061
  • these I plated on CA plates after an hour's rescue
• I transformed iGEM10_005 into lefty pir+ mc1061
  • these I plated on a KC plate after an hour's rescue

Conor McClune 14:03, 8 July 2010 (EDT)

Assembly of iGEM10_007 and iGEM10_013

Restriction Digest of iGEM10_004,iGEM10_005 and iGEM10_011

Samples checked: iGEM10_004#7 ,iGEM10_005#1 and iGEM10_011#1
Well contents:

• 12uL miniprepped DNA
• 2uL BamHI
• 2uL EcoR1
• 1.5 NEB2

incubated at 37 for 1hr Gel Results:

GEM10_004 #7:

• expected sizes:2756,3580
• smeared, but does not look like there are two bands

iGEM10_005 #1:

• expected sizes:2662,2163
• smeared, but it looks like there is an incorrect band around 1kb

iGEM10_011 #1:

• expected sizes:3573,2736
• smeared, second band possibly present, though faint

Analytical PCR of iGEM10_004,iGEM10_005 and iGEM10_011

Well	Sample	Part Checked for	Fwd Oligo	Rev Oligo	Expected Size	Acceptable Band?
1	iGEM10_004 #7	iGEM10_003	ca998	igemTen008	1164	yes, but also a band at 300bp
2	iGEM10_004 #7	Bjh2294	igemTen007	G00101	2607	no
3	iGEM10_005 #1	sbb04	ca998	igemTen004	1888	very faint band
4	iGEM10_005 #1	iGEM10_002	igemTen003	igemTen006	366	no
5	iGEM10_011 #1	iGME10_008	ca998	igemTen020	1157	yes, but also a band at 400bp
6	iGEM10_011 #1	Bjh2294	igemTen019	G00101	2607	no

• Well contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA
• run on program 3K45

Results:

• bands in lanes 1,3,5, but not 2,4,6, indicating that none of the parts were assembled correctly. It looks like there is only the lefty half of each part in these plasmids.

Remake of basic part Sbb10 from 140L stocks

I have not been able to digest it and the Robot Assembly team has never been able to successfully use it in an assembly, so I am going to re-eco/bam it from a BioE 140L stock.

Digest: Well contents: 4uL H20, 4uL stock sbb10, 1.1uL NEB2, 1uL Eco, 1uL Bam. Digested for 1hr.

Ligation: Ligated into pMLL6 using standard procedure.

Transform:Transformed ligation mixture into 100uL jtk030 comp cells (w/ 20uL KCM).

• Rescued for 1.5 hour.
• plated entire culture on KA plate

Conor McClune 15:08, 7 July 2010 (EDT)

2AB assembly of iGEM10_007 and iGEM10_013

Failure of iGEM10_010

Due to all the cotransformations I am getting, I am going to gel purify the digestion fragments

• sbb10:
  • digested with Xhol/BamH1
  • expected sizes: 2633*, 1026
• iGEM10_009:
  • digest with Xhol/BglII
  • expected sizes: 1343*, 1681

Gel Results of yesterdays digestions:

• I gel purified the desired fragment of iGEM10_009, but it seems sbb10 was not digested well, so I will redo the digestion

Digestion

I will be redoing the digestion of sbb10 while also digesting iGEM10_004, iGEM10_005, iGEM10_011 for restriction mapping.

Sbb10

• 3uL H20
• 8uL (1/2 diluted) miniprepped DNA (lefty methylated)
• 1.5uL NEB2 + ATP
• 1uL Xhol
• 1uL BamH1

• the part is still not digested. potential problems:
  • bad enzyme?

Restriction Digests

• created 24uL Eco/Bam MM
• added 6uL MM to 4uL of iGEM10_004 #7, iGEM10_005 #1, iGEM10_011 #1

Results:

iGEM10_004 #7: 2756,3580

• expected sizes:

iGEM10_005 #1:

• expected sizes:2662,2163

iGEM10_011 #1:

• expected sizes:3573,2736

Results

• unclear, though only one band seems visible. I will rerun the digestion with a higher volume of DNA

Conor McClune 17:11, 6 July 2010 (EDT)

Manual Assembly of iGEM10_004 ,iGEM10_005 ,iGEM10_010 and iGEM10_011

Picking Colonies

• picked 8 colonies of each transformation
• set up Col PCR and swiped cotransformation plate

Col PCR results:

• note, all the PCR results seem to be wrong, but many are off by the same amount, so perhaps the ladder is off
  

lanes 1-8:iGEM10_004 - expected size 3771
lanes 10-17: iGEM10_005 - expected size 2354

• correct bands:
  • iGEM10_004: lanes 4-7, with 7 being the strongest
    • 6 is cotransformed
    • miniprep: 4,5,7
  • iGEM10_005: lanes 1-3, 5-8
    • none are cotransformed
    • miniprep 1,2,3

lanes 1-8:iGEM10_010 - expected size 1505

• correct bands:
  • none - 100% cotransformed

lanes 1-8:iGEM10_011 - expected size 3764

• correct bands:
  • lanes 1,3 (neither cotransformed)

Transformation of Bjh2294 to replenish stock

• yesterday's transformation failed, because there were no colonies
• I will redo the transformation today

Procedure:

• add 50uL H20, 30uL KCN and 2uL DNA to 150uL mc1061 competent cells
• keep on ice for 20 min
• heat shock for 90 sec at 42*C
• mixed with 200uL 2YT and rescue for 1hr
• plate on KA plate

Conor McClune 18:14, 5 July 2010 (EDT)

Digestion for Restriction Mapping and Assembly

• made 7uL lefty and righty digestion master mix
• added made double the digestion solution for parts we constructed, so I would be able to run a restriction mapping gel with half:

Well	Part	'	MM	Volume MM
1	sbb04	7-H1	L	7
2	iGEM10_003b		L	14
3	sbb10	22-A3	L	7
4	iGEM10_008d		L	14
5	iGEM10_002d		R	14
6	Bjh2294	24-G10	R	7
7	iGEM10_009b		R	14
8	Bjh2294	24-G10	R	7

• incubated wells at 37 for 1hr
• zymo purified each of the wells
• used one uL of each purified digest for ligation
  • combined wells:
    • 1 with 5 (to make iGEM10_004)
    • 2 with 6(to make iGEM10_005)
    • 3 with 7(to make iGEM10_010)
    • 4 with 8(to make iGEM10_011)

Restriction Digest

• digested with lefty MM (Xhol/BamHI)
  • iGEM10_003b - expected sizes:2671,1064
  • iGEM10_008d - expected sizes:2664,1064
• digested with righty MM (Xhol/BglII)
  • iGEM10_002d - expected sizes:1681, 1430
  • iGEM10_009b - expected sizes:1681, 1343

Results: The strongest bands fit the desired bands. It looks like all these parts are good, though there are some other junk bands.

Retransformation of Bjh2296

• our first stock of Bjh2296 was getting low, so I retransformed it in mc1061
• note: 2YT used for rescue may be contaminated
• plated on KA plate

Conor McClune 14:45, 4 July 2010 (EDT)

2AB construction of iGEM10_007 and iGEM10_013

Results from yesterdays Col PCR of iGEM10_004 and iGEM10_011

• iGEM10_004 - expected size: 3771

• iGEM10_011 - expected size: 3764

Results:
None of the col PCR fragments are the correct size for either of the parts.
Next Steps:

• do restriction digest to make sure the parts I am combining are correct
• redo 2AB assembly

Conor McClune 16:41, 3 July 2010 (EDT)

2AB construction of iGEM10_007 and iGEM10_013

Picking colonies

• picked 8 colonies each for iGEM10_004 and iGEM10_011
• swiped each colongy on a AMp/Cam/Kan plate to check for cotransformation
• ran 4K col pcr

Conor McClune 20:13, 2 July 2010 (EDT)

2AB construction of iGEM10_007 and iGEM10_013

• problem: yesterday I plated the two lefty cultures (containing parts iGEM10_004 and iGEM10_011) on KA plates rather than CA plates. Luckily I had saved the digestions from yesterday, so I religated and retransformed

Picking Colonies

• picked 4 colonies each from the plates containing iGEM10_005 and iGEM10_010
• swiped each colony on a cotransformation plate (Amp) after dipping in col PCR solution and 2uL KC media

Col PCR

• lanes 1-4: iGEM10_010 -expected size is 1500
• lanes 5-8: iGEM10_005 -expected size is 2350

• none of the colonies for either part are the right size. Both colonies need to be repicked
  

Repicking Colonies

• picked 8 more colonies each of iGEM10_010 and iGEM10_005
• ran a col PCR on COL4K
• swiped each colony on an Amp plate to check for cotransformation

Results

• all 8 colonies of iGEM10_010 are cotransformed
• Gel results of col PCR for iGEM10_005 (expected size 2350):

Choano Assays

• on Tuesday(3 days ago), I split choanos 1:15 into choano media under the following conditions:
  • 25*C(control)
  • 37*C incubator
  • 37*C with shaking
  • 1mM NH4
  • 10mM NH4
• results were unconclusive, because even in the control choano density was too low for counts to be reliable, and florescence was too dim to see

Conor McClune 13:34, 1 July 2010 (EDT)

2AB assembly for iGEM10_007 and iGEM10_013

'	Lefty	'	Righty	'	Product
	Part	Version	Part	Version
iGEM10_007
	sbb04	n/a	iGEM10_002	d	iGEM10_004
	iGEM10_003	a	Bjh2294	n/a	iGEM10_005
iGEM10_013
	sbb10	n/a	iGEM10_009	b	iGEM10_010
	iGEM10_008	d	Bjh2294	n/a	iGEM10_011

Digestion

• made up 35uL of lefty and righty MM
• mixed 4 uL of sbb04,iGEM10_003b,sbb10,iGEM10_008d each with 7uL lefty MM
• mixed 4 uL of iGEM10_002d,Bjh2294,iGEM10_009,Bjh2294 each with 7uL righty MM
• incubated for 1hr at 37*C

Ligation

• in each ligation: 6.5uL H2O, 1uL ligation buffer, 0.5uL ligase, 1uL lefty DNA, 1uL righty DNA
• Transformed into 70uL lefty and righty cells (parts 004 and 011 into lefty strain, and parts 005 and 010 into righty strain).
• plated lefty strains on CA and righty strains on KC plates

Analytical PCR of iGEM10_020

• Well contents: 33uL Taq/DMSO MM, 1uL template DNA, 1uL each oligo
• program: 4K55

Well	Part	fwd oligo	reverse oligo	best annealing temp	parts tested for:	expected size
1	iGEM10_020a	ca56	igemTen022	55	igem10_001 and igem10_017	1800
2	iGEM10_020a	igemTen021	G00101	55	igem10_016 and Bjh2294	3500
3	iGEM10_020b	ca56	igemTen022	55	igem10_001 and igem10_017	1800
4	iGEM10_020b	igemTen021	G00101	55	igem10_016 and Bjh2294	3500

Gel Results

• iGEM10_020b is complete