User:GeorgeXu
Important Stuff that was done (in reverse chronological order)
7/27/07
=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)
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 |
T02 0.106 |
T02 0.106 |
J39 0.106 |
J39 0.106 |
J39 0.106 |
S23I07 0.106 |
S23I07 .106 |
S23I07 0.106 |
S23E40 0.105 |
S23E40 0.105 |
S23E40 0.105 |
B | S08E40 .109 |
S08E40 .109 |
S08E40 .109 |
S08I07 0.11 |
S08I07 0.11 |
S08I07 0.11 |
BLANK LB | BLANK LB | BLANK LB | BLANK LB | BLANK LB | BLANK LB |
C | T02 0.002 |
T02 0.002 |
T02 0.002 |
J39 0.001 |
J39 0.001 |
J39 0.001 |
S23I07 0.002 |
S23I07 0.002 |
S23I07 0.002 |
S23E40 0.002 |
S23E40 0.002 |
S23E40 0.002 |
D | S08E40 .003 |
S08E40 .003 |
S08E40 .003 |
S08I07 0.004 |
S08I07 0.004 |
S08I07 0.004 |
BLANK LB | BLANK LB | BLANK LB | BLANK LB | BLANK LB | BLANK LB |
E | T02 0.043 |
T02 0.043 |
T02 0.043 |
J39 0.038 |
J39 0.038 |
J39 0.038 |
S23I07 0.040 |
S23I07 0.040 | S23I07 0.040 |
S23E40 0.041 |
S23E40 0.041 |
S23E40 0.041 |
F | S08E40 .042 |
S08E40 .042 |
S08E40 .042 |
S08I07 0.045 |
S08I07 0.045 |
S08I07 0.045 |
BLANK LB | BLANK LB | BLANK LB | BLANK LB | BLANK LB | 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
Date | Sequence | Result | Comment |
6.27.07 | F1610 | rong | The sequence length is only ~300 bp vs. 798 bp. The sequence contains only the terminator and random junk |
6.27.07 | I13263 | w00t | - |
6.27.07 | I13273 | w00t | - |
7.11.07 | F2620>E0240 | w00t | - |
7.11.07 | I13273 | w00t | - |
7.11.07 | J23039>T9002 | w00t | - |
7.11.07 | S03608>I13507 | w00t | - |
7.11.07 | S03623>E0240 | w00t | - |
7.11.07 | S03623>I13507 | w00t | - |
7.16.07 | R0011 | w00t | - |
7.16.07 | R0011 | w00t | - |
7.16.07 | R0011 | w00t | - |
7.17.07 | R0011<(J23039>T9002) | rong | It is just J23039>T9002 |
7.24.07 | E0240 | w00t | - |
7.24.07 | F2620 | w00t | - |
Miniprepping, Nanodrop, and Sequencing S03608.I13507
I miniprepped and sent S03608>I13507 for sequencing. Elapsed time: 20 minutes.
Colony PCR of various parts
Perry and I colony PCR'ed the following parts:
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.
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:
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.
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
- For switching fluorophores
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:
Growing Liquid Cultures
I grew liquid cultures:
- For RP-JT constructions
- For switching fluorophores
7/22/07
Colony PCR of R-PJT
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 |
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.
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.
7/17/07
Ligation and Transformation of tetR-JT and RFP-JT constructs
I Roche ligated the following:
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
Dephosphorylating the R-P constructs
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 |
I then incubated at 37dC for 1 hour and heat inactivated at 65dC for 10 minutes.
Sequencing the R-P constructs
I prepared and sent off reaction mixes. A summary of the order below:
Label | Construct | Primer |
AV01 | J-hisGFPmek_5_1 | VF2 |
AV02 | J-hisGFPmek_5_1 | VR |
AV03 | J_hisGFPmek_8_1 | VF2 |
AV04 | J_hisGFPmek_8_1 | VR |
AV05 | R0011 <P0340 colony #2 | VF2 |
AV06 | R0011 <P0340 colony #2 | VR |
AV07 | R0051 <P0140 colony #1 | VF2 |
AV08 | R0051 <P0140 colony #1 | VR |
AV09 | R0051 <P0340 colony #1 | VF2 |
AV10 | R0051 <P0340 colony #1 | VR |
AV11 | R0052 <P0140 colony #2 Lower Right | VF2 |
AV12 | R0052 <P0140 colony #2 Lower Right | VR |
AV13 | R0052 <P0340 colony #1 Upper Right | VF2 |
AV14 | R0052 <P0340 colony #1 Upper Right | VR |
Digesting JT and R-P constructs
I digested the following for 99:99 minutes:
Construct | DNA | Enzyme 1 | Enzyme 2 | Buffer 2 | BSA |
R0011 <P0340 colony #2 | 40 µL | 1 µL XbaI | 1 µL PstI | 4.7 µL | .5 µL |
R0051 <P0140 colony #1 | 33 µL | 1 µL SpeI | 1 µL PstI | 4.0 µL | .5 µL |
R0051 <P0340 colony #1 | 35 µL | 1 µL SpeI | 1 µL PstI | 4.2 µL | .5 µL |
R0052 <P0140 colony #2 Lower Right | 30 µL | 1 µL SpeI | 1 µL PstI | 3.6 µL | .5 µL |
R0052 <P0340 colony #1 Upper Right | 33 µL | 1 µL SpeI | 1 µL PstI | 4.0 µL | .5 µL |
J23039 <T9002 | 33 µL | 1 µL SpeI | 1 µL PstI | 4.0 µL | .5 µL |
Miniprep and Nanodrop of JT and the R-P constructs
I miniprepped the following parts:
- R0011 <P0340 colony #2
- R0051 <P0140 colony #1
- R0051 <P0340 colony #1
- R0052 <P0140 colony #2 Lower Right
- R0052 <P0340 colony #1 Upper Right
- J23039 <T9002
- 5
- 8
The "5" and "8" were Perry's samples. Note that the R0052 <P0340 colony #1 Lower Right is missing. That is because the overnight culture didn't seem to grow. After centrifuging several times, there was still no pellet, so I just tossed that sample out.
The Nanodrop results (after Nanodropping, I made the following dilutions for sequencing samples):
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 |
Miniprep Protocol Nanodrop Protocol
7/16/07
Liquid Cultures
I grew up liquid cultures of the following constructs, which come from the colony PCR's that Stephanie did in the morning:
- R0011 <P0340 colony #2
- R0051 <P0140 colony #1
- R0051 <P0340 colony #1
- R0052 <P0140 colony #2 Lower Right
- R0052 <P0340 colony #1 Upper Right
- R0052 <P0340 colony #1 Lower Right
- J23039 <T9002
The Upper/Lower Right is due to the fact that the colony PCR plate had duplicates of those colonies and it was unclear which colony was correct (at least, the PCR and gel was correct). So, I took both samples and labeled one as coming from the upper right of the plate (when viewed with the handwriting going the correct way) versus lower right.
Dephosphorylation of J04450 and JT
I used Antarctive Phosphatase to dephosphorylate the J04450 and the J23039>T9002. I also heat inactivated at 65dC for 15 minutes.
After doing this, I was informed by Perry that I shouldn't dephosphorylate the insert. ARGH! I'll need to regrow, miniprep, and digest the JT.
Sequencing R0011, R0051, R0052
I prepared the R0011, R0051, and R0052 for sequencing. They are AV09, AV10, and AV11 respectively. Stephanie and I dropped them off in the mailbox.
Double Digest of J04450 and JT
I used the following digest mixes:
- J04450
- 25 µL DNA
- 1 µL SpeI
- 1 µL PstI
- 0.5 µL BSA
- 3 µL Buffer 2
- J23039>T9002
- 20 µL DNA
- 1 µL XbaI
- 1 µL PstI
- 0.5 µL BSA
- 2.3 µL Buffer 2
Miniprep and Nanodrop of J04450 and JT
I miniprepped and nanodropped the J04450 and the J23039>T9002.
Nanodrop results:
Miniprep Protocol Nanodrop Protocol
7/15/07
Liquid cultures
I grew up liquid cultures of J04450 and JT in preparation for miniprep, digest, ligation, etc. on Monday 7/16/07.
Transformation of constitutive tetR constructs: Take Two
I transformed the
into Top10 and plated on LB carb plates.
This was done in preparation for making the constitutive tetR+JT construct.
Ligation of P0140, P0340, R0011, and R0051: Take Two
I Roche ligated the following parts:
This was done in preparation for making the constitutive tetR+JT construct.
Vacufuge of R0011 and R0051: Take Two
There were about .3 mL of water left in the the R0011, R0051, and R0052, so I vacufuged for about 2 hours and then resuspended in 20 µL nuclease-free water.
This was done in preparation for making the constitutive tetR+JT construct.
Clonewell of R0011 and R0051: Take Two
I clonewelled the R0011, R0051, and R0052.
This was done in preparation for making the constitutive tetR+JT construct.
Dephosphorylation of R0011 and R0051: Take Two
After discovering that the colony PCR's of the BL21 transformations were horrible, Perry decided we should try it again, but dephosphorylate before we clonewell. So I dephosphorylated the R0011, R0051, and R0052 with the following mix:
- 38 µL DNA
- 3 µL Antarctic Phosphatase
- 4.6 µL Antarctic Phosphatase Buffer
This was done in preparation for making the constitutive tetR+JT construct.
7/13/07
Transformation of constitutive tetR constructs
I transformed the following ligated parts into BL21:
This was done in preparation for making the constitutive tetR+JT construct.
Ligation of P0140, P0340, R0011, and R0051
I ligated the combinations of parts:
with the Roche Ligation Kit (the small white box with the tubes numbered 1-3)
Protocol:
- 3 µL vector (R0051 and R0011) + 7 µL insert (P0140 and P0340)
- Vortex
- 10 µL Vial 1 (ligation buffer)
- Vortex
- 1 µL Vial 3 (ligase)
- Vortex, Spin down
- Let stand for 5 min at room temperature
This was done in preparation for making the constitutive tetR+JT construct.
Dephosphorylation of R0011 and R0051
I treated the R0011 and R0051 with Antarctic Phosphatase. My mix was:
- R0011
- 23.2 µL gel extracted R0011
- 2.7 µL Antarctic Phosphatase Buffer
- 1 µL Antarctic Phosphatase
- R0051
- 22.3 µL gel extract R0051
- 2.6 µL APB
- 1 µL AP
I then incubated for 1 hr @ 37dC and heat inactivated for 10 min @ 65dC.
This was done in preparation for making the constitutive tetR+JT construct.
Vacufuge of P0140, P0340, R0011, and R0051
I vacufuged the samples digested on 7/12/07 for a total of about 3 hours. I then resuspended the samples in about 20 µL of nuclease-free water. I nanodropped them, but the numbers were ridiculously high (~200 ng/µL) and the curves looked awful. Note to self: never Nanodrop gel extraction again.
This was done in preparation for making the constitutive tetR+JT construct.
7/12/07
My First Clonewell
- Get iBase+Clonewell gel. Remove gel (leave the comb!) and place into iBase.
- Pre-run (Mode 0) for two minutes while the comb is still in the gel
- Take out comb, load each well in top row with 20 µL samples, the middle well with ~10 µL ladder, and the bottom row wells with 20 µL nuclease-free water
- Run the gel with (Mode 5: Run Clonewell), watch the bands and stop the gel when your desired band hits the dashed line above the bottom row of wells
- With the gel stopped, fill the bottom row wells with nuclease-free water.
- Run the gel again. When you see the band starting to enter the well, take out the sample in the well and put into clean labeled centrifuge tube. Then refill the well with 20 µL nuclease-free water and continue running the gel.
- Repeat this procedure until you get enough sample. If the band runs past the well, you can use Mode 6: Reverse Clonewell to make the band move back into the well
So basically, I did this for the stuff that was digested (see immediately below) and I ended up with a lot of water, so I am going to vacufuge the results tomorrow.
This was done in preparation for making the constitutive tetR+JT construct.
Digests of P0140, P0340, R0011, and R0051
- XbaI and PstI digests
- SpeI and PstI digest
- Mixture
- 42.5 µL DNA
- 1 µL enzyme 1
- 1 µL enzyme 2
- 0.5 µL BSA
- 5 µL Buffer 2
This was done in preparation for making the constitutive tetR+JT construct.
Perry's E-gel
I ran an E-gel for Perry's colony PCR.
- Take out E-gel (leave comb in) and put in one of the bases. NOTE: One of the red bases has a faulty contact which requires covering by aluminum foil to work correctly.
- Pre-run the gel for 2 minutes. (Press and hold either 15min or 30min on the red bases or Mode 0 on the gray bases)
- When the pre-run is finished, press any button to stop the beeping.
- Load 10 µL of nuclease-free water and 10 µL of ladder into the first well. Then 10 µL nuclease-free water + 10 µL sample into every other well. If there are empty wells, put in 20 µL of nuclease-free water into the empty wells. NOTE: Nick mentioned this and it is a pretty good idea. Put the ladder in the middle well or if there are empty wells, put ladder in the end empty wells. This way, it is easier to compare, especially if the bands bend.
- Run the E-gel for 30-min (Press 30min on the red bases, or run Mode 1 - changing the time if necessary)
- When the run is done, press any button to stop the beeping. Use the UV machine in the small room to visualize the gel.
It confirmed that P0140 and P0340 were correct. However, I don't think his GFPmeks turned out very well.
7/11/07
Sequencing
I sent out the following for Genewiz sequencing:
Part | Nanodrop (ng/µL) |
F2620 > E0240 | 130 |
I13273 | 150 |
J23039 > T9002 | 140 |
S03608 > I13507 | 220 |
S03623 > E0240 | 190 |
S03623 > I13507 | 75 |
The plan was to dilute the above samples to about 40-60 ng/µL and I did the dilutions, but I stupidly ended up using the stock instead of the dilution so our samples had a LOT more DNA than we had planned. Eh, it still ended up working out.
Order Summary:
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 |
7/10/07
7/9/07
Growing Liquid Cultures of Tomorrow's Plate Reader Samples
Stephanie and I grew liquid cultures of the following parts:
Also, Stephanie left the T9002 (+) sample incubating with 1000 nM OHHL to serve as a comparison to J37015.
Colony PCR of Ligated Parts
Stephanie, Perry, and I colony PCR'ed the parts that they ligated the week before.
See Stephanie's notebook for results.
6/29/07 FACS DAY (I13263)!
- 8:30 AM - Stephanie and I took the OD of the overnight cultures.
- Results - 1.785, 1.793, 1.780
- 9:15 AM - Stephanie and I performed first dilution (growing the 4 hr induction cells). Unfortunately, we didn't dilute correctly...
- 1:40 dilution: 50 µL liquid culture + 148 µL LB broth + 2 µL ampicillin
- 1:80 dilution: 25 µL liquid culture + 173 µL LB broth + 2 µL ampicillin
- 1:100 dilution: 20 µL liquid culture + 178 µL LB broth + 2 µL ampicillin
- 10:30 AM - Oopsie, we realized we didn't dilute 1:40, etc.; instead, we had actually diluted 1:4, etc. We measured the OD's of these incorrectly diluted samples and rediluted the samples. Note that in order to measure the OD, we diluted samples 1:100, measured the OD, and multiplied the measured OD by 100 to give us our measurement for the actual sample.
- 1:4 sample OD = 3, redilute 1:100 giving us a sample of about OD=0.03
- 1:8 sample OD = 1.8, redilute 1:30 giving us a sample of about OD=0.05
- 1:10 sample OD = 1.3, redilute 1:100 giving us a sample of about OD=0.01
- Also, we diluted 1:40, 1:80, and 1:100 2 mL samples from the overnight stock to grow as our 2 hr induction cells
- 11:00 AM - I diluted the stock solution 1:10,000 and 1:1,000,000 in 100 µL 200-proof ethanol to give a 97 µM and 97 nM solution.
- 11:30 AM - Stephanie and I diluted 1:40, 1:80, and 1:100 2 mL samples from the overnight stock to grow as our 1 hr induction cells
- 12 noon - Stephanie and I took the OD of the 4 hr induction cells
- 4 hr induction cells, 1:40: 0.2
- 4 hr induction cells, 1:80: 0.2
- 4 hr induction cells, 1:80: 0.1
Since each of these were about 2 mL samples, we didn't have enough sample to have 2 mL cuvette samples. Instead, we used 1mL of cells with 10.5 uL HSL. 2 samples were taken from the 1:40 first dilution (8:30am) - used for the 10 and 100nM HSL runs - and 1 from the 1:80, used with 1nM HSL.
- 1:30 pm - Took OD of the 1hr and 2hr samples + Induce the 2 hr samples
See Stephanie's entry for the rest
Transformation and Liquid Culture of Assorted BioBricks
Perry and I transformed and prepared liquid cultures of the following parts:
6/28/07
Resuspending the HSL stock
I took the 25 mg of N-Butyryl-DL-homoserine lactone and resuspended in 1.5 mL of 200-proof ethanol. I then put the tube in the Quorum sensing box in the freezer at Stephanie's bench.
This was done in preparation for inducing the FACS samples on 6/29/07.
Digestion of F1610: Part Two
Stephanie ran another F1610 digest with
- 2.5 µL Buffer 2
- 0.5 µL BSA
- 0.5 µL XbaI
- 0.5 µL PstI
- 21 µL F1610
Basically it is the same as the previous digestion except we now have 21 µL vs. 15 or 6 µL of DNA. This was done because the Clonewell of the F1610 that Perry and I ran had an almost invisibly faint band corresponding to the F1610. Where did all the DNA go?
Grow Liquid Cultures of I13263
Stephanie and I grew I13263 in preparation for the FACS on 6/29/07.
Sequences
Perry checked the sequences that we got back from Genewiz
Results
- F1610 (something is very wrong...)
- The actual part is 798 bp, we got back a sequence ~300 bp
- The sequence contains the terminator and some random junk after it
- I13263 (w00t)
- The first and last ~650 bp are perfect.
- I13273
- !!!!add something here
!!!!upload the sequences?
Clonewell of F1610
[User:GeorgeXu#My_First_Clonewell Protocol]
Results
F1610 is about 800 bp. There is an almost invisibly faint band at 800 bp. What happened to all the DNA? We need to run it again
Transformation of Additional Parts
Perry and I transformed the following parts
Digestion of F1610 (XbaI, PstI)
Mix 1:
- 2.5 µL Buffer 2
- 0.5 µL BSA
- 0.5 µL XbaI
- 0.5 µL PstI
- 15 µL F1610 (168 ng/µL)
- 6 µL Nuclease-free water
Mix 2:
- 2.5 µL Buffer 2
- 0.5 µL BSA
- 0.5 µL XbaI
- 0.5 µL PstI
- 15 µL Nuclease-free water
- 6 µL F1610 (168 ng/µL)
Perry and I ran two digests of F1610 with different amounts of DNA. The plan is to gel extract and ligate with the promoters that Perry has transformed.
6/27/07
Sequencing the Midiprepped QS parts
Just checking if the Midiprepped sequences are correct. Details of the order:
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 |
Transformation of I13263
Stephanie and I transformed 1 µL of the I3263 that we midiprepped into 30 µL of BL21. We then plated them onto agar+ampicillin plates. We eventually want to carry out a test experiment by inducing the I3263 with different concentrations of HSL to test and possibly start characterizing the part.
Hispeed! Midiprepping of the QS parts
Perry and I Midiprepped the QS parts in order to get DNA that we could sequence and transform.
6/26/07
Growth of QS parts in Liquid Cultures
We grew the QS parts in a liquid LB+ampicillin broth.
Nanodrop
Results
10mer: 389.8 ng/µL
15mer: 278.6 ng/µL
20mer: 168.7 ng/µL
Nucleotide Removal of the 10mer, 15mer, and 20mer library
Again, this was done in preparation for the ligation reaction that we will do with the massive amounts of DNA.
PCR Extension of the 10mer, 15mer, and 20mer library
Basically, this was done in preparation for the ligation reaction that we will do with the massive amounts of DNA.
6/25/07
Transforming and Plating the QS parts
Perry transformed the parts F1610, I13263, and I13272 into Top10 cells and plated them on LB+ampicillin plates. We want to sequence the parts, test them, and eventually use them in our BL21 cells.
6/22/07
Counting colonies: Overnight vs. Short Ligation
We counted the number of colonies in the Overnight vs. Short Ligation plates.
Results
Nanodrop of Ligated Colonies
Results
OmpA1+random library+extension: 50.8 ng/µL
OmpA2+random library+extension: 50.7 ng/µL
The curves looked smooth. %
6/21/07
Protein Gel of OmpA+His/OmpA+Strep Cells
We ran this protein gel to see if the OmpA1+His, OmpA1+Strep, OmpA2+His, and OmpA2+Strep was actually expressed. Also, we wanted to see if there was any difference between the immediate induction, delayed induction, and no induction.
Bacterial Innoculation and Induction
Inocculation and Induction Protocol
Measuring Optical Density Protocol
We took the OmpA1+His, OmpA1+Strep, OmpA2+His, and OmpA2+strep that were grown in liquid culture overnight starting on 6/20/07 and split them into three sets. The first set was not induced, the second set was immediately induced with 10 µL IPTG¸ and the final set was induced once it hit log phase, which we determined by measuring the optical density.
6/20/07
Gel Extraction
We extracted the DNA from the gel we ran on 6/19/07
6/19/07
Running the OmpA1/OmpA2 gel
We poured a gel and ran the DNA from the dephosphorylated samples.
Dephosphorylating the OmpA1/OmpA2
Six times this amount of mix was made as a Master Mix in order to dephosphorylate the OmpA1 and OmpA2. There were five groups total:
- Group 1: OmpA1, Nhe1, Pst1
- Group 2: OmpA1, Nhe1, Pst1
- Group 3: OmpA2, Nhe1, Pst1
- Group 4: OmpA2, Nhe1, Pst1
- Group 5: Perry...