User:GeorgeXu: Difference between revisions

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==Important Stuff that was done (in reverse chronological order)==
==Important Stuff that was done (in reverse chronological order)==
===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)
*[http://parts.mit.edu/registry/index.php/Part:BBa_B0015 B0015] EX
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0140 P0140] colony #1
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0051 R0051] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 P0340] colony #1
*[http://parts.mit.edu/registry/index.php/Part:BBa_R0011 R0011] <[http://parts.mit.edu/registry/index.php/Part:BBa_P0340 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===
===7/20/07===
====Clonewell and Vacufuging the RP and JT parts====
====Clonewell and Vacufuging the RP and JT parts====

Revision as of 14:24, 21 July 2007

Important Stuff that was done (in reverse chronological order)

7/21/07

Clonewell RP and JT

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

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.

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:

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

Protocol

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.

Protocol

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:

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:

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.

Protocol

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

Protocol

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.

Protocol

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.

Protocol

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.

Protocol

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.

Protocol

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.

Protocol

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:

  1. 3 µL vector (R0051 and R0011) + 7 µL insert (P0140 and P0340)
  2. Vortex
  3. 10 µL Vial 1 (ligation buffer)
  4. Vortex
  5. 1 µL Vial 3 (ligase)
  6. Vortex, Spin down
  7. Let stand for 5 min at room temperature

This was done in preparation for making the constitutive tetR+JT construct.

Protocol

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.

Protocol

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

  1. Get iBase+Clonewell gel. Remove gel (leave the comb!) and place into iBase.
  2. Pre-run (Mode 0) for two minutes while the comb is still in the gel
  3. 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
  4. 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
  5. With the gel stopped, fill the bottom row wells with nuclease-free water.
  6. 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.
  7. 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.

Protocol

Perry's E-gel

I ran an E-gel for Perry's colony PCR.

  1. 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.
  2. 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)
  3. When the pre-run is finished, press any button to stop the beeping.
  4. 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.
  5. Run the E-gel for 30-min (Press 30min on the red bases, or run Mode 1 - changing the time if necessary)
  6. 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.

Results

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.

Protocol

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

Colony PCRs

7/9/07

Growing Liquid Cultures of Tomorrow's Plate Reader Samples

Protocol

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

Protocol

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)!

FACS-related

  • 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

Transformation Protocol

Liquid Cultures Protocol

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

  1. 2.5 µL Buffer 2
  2. 0.5 µL BSA
  3. 0.5 µL XbaI
  4. 0.5 µL PstI
  5. 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

Protocol

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

Protocol

Perry and I transformed the following parts

Digestion of F1610 (XbaI, PstI)

Mix 1:

  1. 2.5 µL Buffer 2
  2. 0.5 µL BSA
  3. 0.5 µL XbaI
  4. 0.5 µL PstI
  5. 15 µL F1610 (168 ng/µL)
  6. 6 µL Nuclease-free water

Mix 2:

  1. 2.5 µL Buffer 2
  2. 0.5 µL BSA
  3. 0.5 µL XbaI
  4. 0.5 µL PstI
  5. 15 µL Nuclease-free water
  6. 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.

Protocol

6/27/07

Sequencing the Midiprepped QS parts

Protocol

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

Protocol

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

Protocol

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

Protocol

We grew the QS parts in a liquid LB+ampicillin broth.

Nanodrop

Protocol

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.

Protocol

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.

Protocol

6/25/07

Transforming and Plating the QS parts

Protocol

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

Protocol

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

Protocol

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.

Results

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.

Optical Density Results

6/20/07

Gel Extraction

Protocol

We extracted the DNA from the gel we ran on 6/19/07

Gel after Excision

6/19/07

Running the OmpA1/OmpA2 gel

Protocol

We poured a gel and ran the DNA from the dephosphorylated samples.

Results

Dephosphorylating the OmpA1/OmpA2

Protocol

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:

  1. Group 1: OmpA1, Nhe1, Pst1
  2. Group 2: OmpA1, Nhe1, Pst1
  3. Group 3: OmpA2, Nhe1, Pst1
  4. Group 4: OmpA2, Nhe1, Pst1
  5. Group 5: Perry...
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