User:GeorgeXu

Important Stuff that was done (in reverse chronological order)

9/17/07

Double Digest of AIDA-1 and PCRed rdm-lib

Reagent	Concentration	Volume	Total in soln
AIDA-1/pet29b+ DNA	~150 ng/uL	23.5 uL	~3.5 mg
NotI (NEB)	10 U/uL	2 uL	10 U
NheI (NEB)	10 U/uL	1 uL	10 U
Buffer 2	10x	5 uL	1x
BSA	1000x	.5 uL	10x
DEPC water	-	18 uL	-

Reagent	Concentration	Volume	Total in soln
PCRed insert	-	21 uL	-
NheI (NEB)	10 U/uL	2.5 uL	10 U
NotI (NEB)	10 U/uL	5 uL
Buffer 2	10x	5 uL	1x
BSA	1000x	.5 uL	10x
DEPC water	-	16 uL	-

Sequential Digest of AIDA-1 and PCRed rdm-lib

Reagent	Concentration	Volume	Total in soln
AIDA-1/pet29b+ DNA	~150 ng/uL	23.5 uL	~3.5 mg
NheI (NEB)	10 U/uL	1 uL	10 U
Buffer 2	10x	5 uL	1x
BSA	1000x	.5 uL	10x
DEPC water	-	20 uL	-

Reagent	Concentration	Volume	Total in soln
PCRed insert	-	21 uL	-
NheI (NEB)	10 U/uL	2.5 uL	10 U
Buffer 2	10x	5 uL	1x
BSA	1000x	.5 uL	10x
DEPC water	-	21 uL	-

9/15/07

Miniprepping AIDA-1/pet29b+

Miniprepped the AIDA-1 (~150 ng/uL for all three samples; curves looked good)

PCRing Random Library

Realized we don't have NotI enzyme so set up a PCR reaction for the random library + AIDA:

Reagent	Concentration	Volume
AIDA-1/pet29b+ DNA	~1.5 ng/uL	.5 uL
Primer 1 (MSN27-strong)	50 uM	3 uL
Primer 2 (MNSIGEM2)	100 uM	1.5 uL
Platinum PCR supermix	-	45 uL

Both primers can be found in the -20dC freezer in the small room in a box labeled with green tape (something like "Mike Strong Plasmids for iGEM 2007"). These are primers for the 15mer library.

9/14/07

Growing Liquid Cultures (AIDA-1/pet29b+)

Grew up 3x4.5mL of AIDA-1 in LB kan from Perry's frozen stock (loc:Perry's box in the -80dC). Tubes were labeled something like "AIDA-1/pet29b+."

8/18/07

Rdm lib + AIDA construction

AP treated 50 µL of AIDA1 cut with NotI/NheI in pet29 with 6 µL AP buffer and 4 µL AP
Clonewelled the samples
Vacufuged and PCR purified the samples
Nanodropped and set up three ligation reactions
1. PCR digest 1 + cut AIDA
  1. PCR digest - 30 µL of 33.8 ng/µL lyophilized and resuspended in 13.5 µL, used 7 µL
  2. cut AIDA - 47 µL of 37.3 ng/µL lyophilized and resuspended in 9 µL, used 3 µL
2. PCR digest 2 + cut AIDA
  1. PCR digest - 32 µL of 45.1 ng/µL lyophilized and resuspended in 19.4 µL, used 7 µL
  2. cut AIDA - 47 µL of 37.3 ng/µL lyophilized and resuspended in 9 µL, used 3 µL
3. PCR digest 3 + cut AIDA
  1. PCR digest - 32 µL of 45.7 ng/µL lyophilized and resuspended in 19.4 µL, used 7 µL
  2. cut AIDA - 47 µL of 37.3 ng/µL lyophilized and resuspended in 9 µL, used 3 µL

Re-electroporating tagged Single Cells + T02 his

I had to miniprep more JT because we used up the plasmid preps from last time

JT DNA into AIDA his cells (from Steph's fridge liquid cultures)
JT DNA into AIDA+strep cells (from Steph's fridge liquid cultures)
RACE DNA into AIDA+strep cells (from Steph's fridge liquid cultures)
RACE DNA into AIDA+strep cells (from Steph's fridge liquid cultures)
AIDA+his DNA (from Mike's iGEM box) into T02 cells

Plates should be in incubator

Checking Tagged Receivers

Round 1
- Colony PCRed the RAE+his, RAE+strep, and T02+strep (the T02+his plate was empty).
- Unfortunately, there are only SybrSafe E-gels left, so no gel picture. Gel summary below (basically RAE+strep 2 and T02+strep 2-5 worked)

Well	Contents	Band Length	Actual Length	Correct?
1	1 kb+	-	-	-
2	RAE+his 1	1000	2000	No
3	RAE+his 2	none	2000	No
4	RAE+his 3	none	2000	No
5	RAE+strep 1	none	2000	No
6	RAE+strep 2	2000	2000	Yes
7	RAE+strep 3	none	2000	No
8	T02+strep 1	1100ish	2000	No
9	T02+strep 2	none	2000	No
10	T02+strep 3	2000	2000	Yes
11	T02+strep 4	2000	2000	Yes
12	T02+strep 5	2000	2000	Yes

Round 2
- Basically I redid RAE+his 1-3 and then streaked three more colonies to get RAE+his4-6. I also checked RAE+strep 2 and T02+strep 3-5 with the T7 promoter/terminator primers. Note I have two wells of RAE+strep 2 because I distrust (is that a word?) colony PCR.
- Results were good. The RAE_strep 2 and T02 strep3-5 check out completely. It appears that RAE+his 3 also checks out. I've set up the T7 PCR reaction for that have it sitting in the black PCR machine on Stephanie's bench.

Well	Contents	Band Length	Actual Length	Correct?
1	1 kb+	-	-	-
2	RAE+his 1	450 (faint)	2000	No
3	RAE+his 2	450 (faint)	2000	No
4	RAE+his 3	2000	2000	Yes
5	RAE+his 4	none	2000	No
6	RAE+his 5	none	2000	No
7	RAE+his 6	none	2000	No
8	RAE+strep 2A	1700ish	1500	Yes
9	RAE+strep 2B	1700ish	1500	Yes
10	T02+strep 3	1700ish	1500	Yes
11	T02+strep 4	1700ish	1500	Yes
12	T02+strep 5	1700ish	1500	Yes

Other

Finally streaked those liquid cultures of the constructs we sequenced.
Streaked the liquid culture of pAC-luxGFP (Voigt construct)
miniprepped the pAC-luxGFP
poured several LBamp+kan plates. They should be labeled with black sharpie on the broad circular face of the plate.

8/14/07

Colony PCR of RACE (new)

RACE #1-3 are correct.

Gel

8/13/07

Fluorescent Microscopy of RACE

I picked from six samples from three plates of RACE and dipped into 10 µL of PBS:

RACE 37dC (new) - the new RACE transformation plate
- Center of the colony (fluorescent)
- Edge of the colony (nonfluorescent)
RACE 37dC (old) - the RACE plate the old transformation
- fluorescent cells
- nonfluorescent cells
RACE - the original RACE transformation plate
- Center of the colony (fluorescent)
- Edge of the colony (nonfluorescent)

Microscopy Results:

For the most part, the expected nonfluorescent cells are nonfluorescent and the fluorescent cells are fluorescent. However, it seems that the original plates are exhibiting stronger fluorescence than the new transformation plates which is opposite of what Nick, Harris, Bill and I observed in the transformation plates
I need to fix my protocol for preparing slides because I have problems with the ends of my slides drying in and some of my samples were very dilute.
These RACE cells didn't have the filament problems like the last RACE I grew. I'm going to retry the RACE+OHHL and RACE overnight to see if I can get some positive results.

Fluorescent Microscopy Pics

8/10/07

Colony PCR of JT + confirm Master Plate

JT's are whacko, the Master Plate constructs are correct. We need to re-transform JT into BL21.

Gel

Frozen Stock Part 1

Perry and I made frozen stocks of some stuff.

8/09/07

Mixed Cultures with same OD

I had a culture of S23I07 diluted to OD=0.057 and a culture of RAE diluted to an OD=0.059. I made a 1:1, 10:1, and 100:1 combined culture (with more of the RAE). I also had a culture of RACE that I induced with ~1 mM.

I grew these for about three hours and then checked them in the plate reader and the fluorescent microscope.

Plate Reader Data 1
Sample	OD	GFP	RFP
RACE+OHHL	0.716	79.196	179.94
1:1	0.866	161.96	257.00
10:1	0.729	314.73	204.50
100:1	0.405	477.33	216.94

Fluorescent Microscope Results #1:

Not too much to say here, it seems that the cells haven't grown enough because we don't see much fluorescene yet and the OD's are still pretty low
One thing of note: the RACE is abnormally filamentous. Something is wrong...

Fluorescent Microscopy Pics 1

I grew the samples for another two hours and then checked them again in the plate reader and the fluorescent microscope.

Plate Reader Data 2
Sample	OD	GFP	RFP
RACE+OHHL	1.235	80.495	165.18
1:1	1.278	205.86	444.09
10:1	1.234	414.76	537.42
100:1	1.047	670.86	234.11

Fluorescent Microscope Results #2:

The RFP fades RIDICULOUSLY fast. The fluorescence fades to black in a couple of seconds.
The GFP in the mixed cultures is pretty good, the RFP is almost invisible. This could be the result of the RFP folding time and the fact that the RFP fades RIDICULOUSLY fast. Seriously, like, Nick and I basically had to say "1...2...3... GO!" to make sure we got the picture on time

Fluorescent Microscopy Pics 2

8/08/07

I took plate reader data of the swab inoculated liquid cultures:

Sample	Spectro. OD	Plate OD	GFP	RFP
S23.I07+R62.A20.E40	2.293	2.047	249.34	5706.1
B15	1.782	1.567	108.47	158.64
S23.I07+T02	2.171	1.956	616.05	6045.8
OHHL+T02	1.812	1.76	263.01	168.93
I22	2.069	1.919	10101	187.39
OHHL+R62.A20.E40	1.923	1.82	1770.6	163.89
R62.A20.E40	1.89	1.735	166.23	175.59
Blank LB	-	0.096	77.822	165.43

I also checked the cultures under the fluorescent microscope:

The S23I + RAE sample showed a lot of red colonies and a very small number of green colonies.
- Maybe the red colonies are just much better competitors? It could also be because I didn't start with equal OD's. I'm going to redo this tomorrow.
The RAE works surprisingly well. By itself, it has a pretty good OFF and the induced with OHHL was brightly fluorescent.

Fluorescent Microscopy Pics

8/07/07

Colony PCR for Master Plate

Colony PCR is a stupid protocol

Gel

8/06/07

Colony PCR for Master Plate

Whaaa?

Gel

8/03/07

Colony PCR of luxpR Senders and Receivers

Whaaa?

Gel

8/02/07

Colony PCR of luxpR JT + mCherry parts

The mCherry colonies were not red T_T.

Gel

8/01/07

Colony PCR of mCherry Part

Most of the streaks looked good

Gel

7/31/07

Colony PCR of luxpR Round 2 Take 1

Colony PCR'ed the transformation plates from last night. The transformation plates looked very good, with lots of colonies.

Gel

7/30/07

luxpR Construction Round 2

Miniprep, Digest, Dephosphorylate, Clonewell, Ligate, Transform:

R0062<A340620
C0061<E0240

The only difference is that the Minipreps turned out a lot better, giving nanodrop values of >130 ng/µL. Also, I stupidly transformed into Top10F' instead of Top10.

7/29/07

Colony PCR of luxpR Round 1 Take 2

I colony PCR'ed the colonies from the transformation plates from last night. The plates were a little better, with around 10 colonies each. I only got *i think* the C0061<I13504 to work. Also, the five bands that should be R0062<A340620 are all about the size of R0062 so I'm going to redo the construction tomorrow.

Gel

7/28/07

Colony PCR of luxpR Round 1 Take 1

Unfortunately, there were like no colonies. Meaning:

R0062<A340620
- Two colonies
C0061<I13504
- Zero colonies
C0061<E0240
- Two colonies

So I colony PCR'ed them and then froze the result (no gel running today, folks).

Religating and Transforming

I re-ligated and transformed some leftover DNA from last night:

Finally, I transformed the constructs into Top10 cells.

7/27/07

Clonewell, Vacufuge, Ligate, Transform

I clonewelled, vacufuged, and then ligated the following:

Finally, I transformed the constructs into BL21 cells.

Dephosphorylating the Vectors

I dephosphorylated the R0062 and C0061 with Antarctic Phosphotase and then heat inactivated them for 10m at 65dC.

Digesting for Double luxpR

I digested the following for 99:99 at 37dC:

Part	DNA	Enzyme 1	Enyzme 2	Buffer 2	BSA
R0062	20 µL	1 µL SpeI	1 µL PstI	2.5 µL	0.5 µL
A340620	20 µL	1 µL XbaI	1 µL PstI	2.5 µL	0.5 µL
C0261	20 µL	1 µL SpeI	1 µL PstI	2.5 µL	0.5 µL
E0240	20 µL	1 µL XbaI	1 µL PstI	2.5 µL	0.5 µL
I13504	20 µL	1 µL XbaI	1 µL PstI	2.5 µL	0.5 µL

Sequencing the C0261

I prepared and sent C0261 for sequencing.

Miniprep for Double luxpR

Perry and I miniprepped:

in order to build the luxpR construct.

7/26/07

FACS

Perry and I diluted down overnight samples and grew them in an incushaker from 9:30-12:30. The numbers indicate OD right before we resuspended in PBS. We tried to resuspend so that the final OD was around 0.003.

JT
- 1:10 (.555)
- 1:100 (.141)
- 1:1000 (.02)
- 1:10000 (.000-.001)
T02&S23I07 1:1000 (.03)
T02&J23039 1:1000 (.03)
F20I07&S23E40 1:1000 (.075)
J04450 1:100 (.064)
I13522 1:100 (.62)
B0015 1:100 (.061)

Media:072707facsgx+pt.zip

Overnight Plate Reader Growth Curve Results

I set up a plate reader as follows. The numbers after the label indicate starting OD. Note: Our plate reader seems to have trouble reading OD's around 0.1, so I'm not completely sure if the 0.1 values are accurate. I took the lower OD readings in the OD machine in the small room, so I'm pretty confident about those.

1

2

3

4

5

6

7

8

9

10

11

12

A

T02
0.106

J39
0.106

S23I07
0.106

S23I07
.106

S23I07
0.106

S23E40
0.105

B

S08E40
.109

S08I07
0.11

BLANK LB

C

T02
0.002

J39
0.001

S23I07
0.002

S23E40
0.002

D

S08E40
.003

S08I07
0.004

BLANK LB

E

T02
0.043

J39
0.038

S23I07
0.040

S23I07 0.040

S23I07
0.040

S23E40
0.041

F

S08E40
.042

S08I07
0.045

BLANK LB

Results:

Starting at OD~0.1 or OD~0.04 it took the samples around 2 1/2 hrs to read OD~0.3 and around a total of 6 hrs to hit stationary phase
Starting at OD~0.003, it took the samples around 4 hours to reach OD~0.3 and around a total of 7 hours to hit stationary phase
There didn't seem to be a big difference between starting at OD~0.1 vs OD~0.04. Of course, this could be due to the fact that I took the OD reading for the OD~0.1 samples in the plate reader, which is inaccurate in that range.
Overall, the samples grew at pretty much the same rate. There appears to be at most a 45-60 min lag between different constructs, but this could be due to variations in starting OD.
The blank LB samples were interesting. They were pretty much flat and low until around 8 hrs in, then they grew exponentially to around OD~1. This seems to indicate that there were very low levels of contamination in these LB wells. I added LB+amp to these wells, so the contamination is probably coming from our own constructs. I don't know if this contamination was present when I loaded the wells or somehow there was cross-well contamination during the reading period.

For all the graphs+raw data: Media:072607platereadergx.xls

7/25/07

Sorting through Sequences

I went through all the sequences we have done and compiled the results

Sequencing Results

Miniprepping, Nanodrop, and Sequencing S03608.I13507

I miniprepped and sent S03608>I13507 for sequencing. Elapsed time: 20 minutes.

Sequencing Results

Colony PCR of various parts

Perry and I colony PCR'ed the following parts:

Gel

7/24/07

Liquid Cultures

Perry and I grew liquid cultures of several things:

Mike's Agar Plate Experiment

Perry and I carried out a preliminary test of Mike's Plate Reader Experiment. Protocol:

Pipet 200 µL of overnight cultures of T9002 and spread onto LB agar plates.
Let the plates sit at room temperature to allow the bacteria to soak in. (We let it sit for about 20 minutes)
Pipet in 10 µL of one of each of the following into the middle of each plate.
1. Overnight culture of S03623 > I13507
2. Overnight culture of S03623 > I13507
3. Overnight culture of S03608 > I13507
4. Overnight culture of S03608 > I13507
5. 2 µM OHHL

We did duplicates because we took from two different overnight cultures, one that was obviously red and one that was not obviously red.

Ligating and Transforming the RP-JT and FluorSwitch

Perry and I performed the following ligations and transformations:

F2620 >I13507

into BL21

We decided not to do the other ligations and transformations because we found out that we already have a S03623 <E0240 and we were colony PCR'ing a S03608<E0240. Finally, because the J23039 >T9002 miniprep and clonewell sucked, we were reluctant to move forward on that ligation.

Clonewell for RP-JT and FluorSwitch

I clonewelled and vacufuged all the digests I did earlier in the day. The band for J23039 >T9002 was almost invisible.

Sequencing the F2620 and E0240

I prepared a sequencing order for F2620 and E0240.

Sequencing Results

Dephosphorylate for RP-JT and FluorSwitch

I dephosphorylated the following:

with Antarctic Phosphatase for 1 hr at 37dC and then heat inactivated for 10m at 65dC.

Digest for RP-JT and FluorSwitch

I digested the following for 99m99s.

Part	DNA	Enzyme 1	Enzyme 2	Buffer 2	BSA
R0011 <P0140	21 µL	1 µL EcoRI	1 µL XbaI	2.5 µL	0.5 µL
R0051 <P0140	21 µL	1 µL EcoRI	1 µL SpeI	2.5 µL	0.5 µL
J23039 >T9002	21 µL	1 µL EcoRI	1 µL SpeI	2.5 µL	0.5 µL
S03623	21 µL	1 µL XbaI	1 µL PstI	2.5 µL	0.5 µL
S03608	21 µL	1 µL XbaI	1 µL PstI	2.5 µL	0.5 µL
E0240	21 µL	1 µL SpeI	1 µL PstI	2.5 µL	0.5 µL
F2620	21 µL	1 µL EcoRI	1 µL SpeI	2.5 µL	0.5 µL
I13507	21 µL	1 µL EcoRI	1 µL XbaI	2.5 µL	0.5 µL

Miniprep (RPJT+FluorSwitch)

I miniprepped the samples that were grown last night:

For RP-JT constructions
- R0011 <P0140
- R0051 <P0140
- J23039 >T9002
For switching fluorophores
- S03623
- S03608
- E0240
- I13507

All the nanodrop data was good (about 150 ng/µL with excellent curves), except for the J23039 >T9002 (50ng/µL)

7/23/07

Colony PCR of RP

I colony PCR'ed the R-P constructs that didn't work out the first time:

Gel

Growing Liquid Cultures

I grew liquid cultures:

For RP-JT constructions
- R0011 <P0140
- R0051 <P0140
- J23039 >T9002
For switching fluorophores
- S03623
- S03608
- E0240
- I13507

7/22/07

Colony PCR of R-PJT

Gel

7/21/07

Clonewell RP and JT

I clonewelled the following things (some are Perry's samples):

1+2B XP
JsLO ES (very faint)
JsLO SP (very faint)
B0015 EX
R0051 <P0140 colony #1
R0051 <P0340 colony #1
R0011 <P0340 colony #2

and then vacufuged them.

Dephosphorylating JT

I dephosphorylated JT with Antarctic Phosphotase (1 hr 37dC) and then heat inactivated at 65dC for 10 min.

7/20/07

Clonewell and Vacufuging the RP and JT parts

I clonewelled the leftovers from the RP and JT digests and then vacufuged and resuspended in 20 µL of nuclease free water. This was done in order to try rebuilding the constitutive tetR+JT construct.

7/19/07

Liquid Cultures of 2 cell system and RFP+JT

I grew liquid cultures of the 2 cell system and the RFP+JT in preparation for plate reader experiments tomorrow.

Sequencing the RFP+JT part

I prepared the RFP+JT part and sent it for sequencing.

Label	Sample	Primer
AV01	J04450-JT	VF2
AV02	J04450-JT	VR
AV03	jSLOPDZ2B	VF2
AV04	jSLOPDZ2B	VR
AV05	jSLOPDZ1B	VF2
AV06	jSLOPDZ1B	VR
AV07	JhisGFPmek	VF2
AV08	JhisGFPmek	VR

Sequencing Results

Colony PCR of the tetR+JT constructs Part II

I chose only non-fluorescent colonies (by checking the colonies under the fluorescent microscope) for this colony PCR.

Results: None of the colony PCR's gave the correct size DNA piece.

Gel

Miniprep the RFP+JT

I miniprepped the RFP+JT sample (originally for transformation in Top10 because BL21 expresses LacI, but we decided that we liked the idea of inducible RFP).

7/18/07

Liquid Cultures

I grew liquid cultures of the "correct" RFP+JT colony resulting from the colony PCR.

Colony PCR of the tetR+JT and RFP+JT constructs

I colony PCR'ed the teR+JT and RFP+JT constructs.

Results: Only one colony seemed correct. It was one of the constitutive RFP+JT parts.

Gel

7/17/07

Ligation and Transformation of tetR-JT and RFP-JT constructs

I Roche ligated the following:

(R0051 <P0140) <(J23039 <T9002)
J04450 <(J23039 <T9002)

and then transformed them into BL21.

Ligation Protocol Transformation Protocol

Clonewell and Vacufuge of the R-P constructs

I clonewelled and vacufuged the R-P constructs and the JT. Unfortunately, I forgot to include the JT with the initial clonewell, so I ended up having to fish the very nasty clonewell out of the garbage (there was a piece of gel on it and chunks of gel were stuck in some of the wells). I used the last two wells (which were thankfully somewhat clean) for ladder and the JT. Unfortunately I ran into a few problems. First, the JT is about 2800 bp and the plasmid is about 2000 bp. The two bands on the clonewell were pretty close and it was difficult to separate the two bands sometimes. Second, the gel stopped running after a while. I noticed bubbles forming along the edges of the gel and whenver I pressed "Go," the gel would make an unhappy beeping noise and blink yellow, so I just stopped running the clonewell

Construct	DNA	AP Buffer	AP
R0011 <P0340 colony #2	40 µL	4.7 µL	2 µL
R0051 <P0140 colony #1	42 µL	4.9 µL	2 µL
R0051 <P0340 colony #1	36 µL	4.2 µL	2 µL
R0052 <P0140 colony #2 Lower Right	40 µL	4.7 µL	2 µL
R0052 <P0340 colony #1 Upper Right	40 µL	4.7 µL	2 µL

Construct	Concentration	Dilution	µL of DNA	µL of Water
R0011 <P0340 colony #2	97.6 ng/µL	1 in 2	10	10
R0051 <P0140 colony #1	173.2 ng/µL	1 in 4	5	15
R0051 <P0340 colony #1	110.3 ng/µL	1 in 2	10	10
R0052 <P0140 colony #2 Lower Right	133.7 ng/µL	1 in 3	7	14
R0052 <P0340 colony #1 Upper Right	132.8 ng/µL	1 in 3	7	14
J23039 <T9002	98.8 ng/µL	none	-	-
5	112.1 ng/µL	1 in 2	10	10
8	148.9 ng/µL	1 in 3	7	14

Part	Nanodrop (ng/µL)
F2620 > E0240	130
I13273	150
J23039 > T9002	140
S03608 > I13507	220
S03623 > E0240	190
S03623 > I13507	75

Tube Label	Part	Primer
AV01	S03623 > I13507	VF2
AV02	S03608 > I13507	VF2
AV03	S03623 > E0240	VF2
AV04	F2620 > E0240	VF2
AV05	I13273	VF2
AV06	J23039 > T9002	VF2
AV07	S03623 > I13507	VR
AV08	S03608 > I13507	VR
AV09	S03623 > E0240	VR
AV10	F2620 > E0240	VR
AV11	I13273	VR
AV12	J23039 > T9002	VR

Tube Label	DNA Name	DNA Length	Primer
AV01	F1610	3987	VF2
AV02	F1610	3987	VR
AV03	I13263	4123	VF2
AV04	I13263	4123	VR
AV05	I13272	4162	VF2
AV06	I13272	4162	VR