User:GeorgeXu: Difference between revisions
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Stephanie and I grew liquid cultures of the following parts: | Stephanie and I grew liquid cultures of the following parts: | ||
*I13263 | *[http://parts.mit.edu/registry/index.php/Part:BBa_I13263|I13263] | ||
*I13273 | *[http://parts.mit.edu/registry/index.php/Part:BBa_I13273|I13273] | ||
*I5311 | *[http://parts.mit.edu/registry/index.php/Part:BBa_I5311|I5311] | ||
*I13522 | *[http://parts.mit.edu/registry/index.php/Part:BBa_I13522|I13522] | ||
*B0015 | *[http://parts.mit.edu/registry/index.php/Part:BBa_B0015|B0015] | ||
*T9002 (+) | *[http://parts.mit.edu/registry/index.php/Part:BBa_T9002|T9002 (+)] | ||
*T9002 (-) | *[http://parts.mit.edu/registry/index.php/Part:BBa_T9002|T9002 (-)] | ||
Also, Stephanie left the T9002 (+) sample incubating with 1000 nM OHHL to serve as a comparison to J37015. | Also, Stephanie left the T9002 (+) sample incubating with 1000 nM OHHL to serve as a comparison to J37015. | ||
====Colony PCR of Ligated Parts==== | ====Colony PCR of Ligated Parts==== | ||
[[IGEM:Harvard/2007/Protocols#Colony_PCR.2Fplate_streak|Protocol]] | [[IGEM:Harvard/2007/Protocols#Colony_PCR.2Fplate_streak|Protocol]] |
Revision as of 12:24, 10 July 2007
Me
Important Stuff that was done (in reverse chronological order)
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.
- I13272
- !!!!add something here
!!!!upload the sequences?
Clonewell of F1610
!!!!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
!!!!descriptions
- R0040
- Promoter
- R0051
- Promoter
- R0011
- Promoter
- E0240
- RBS+GFP+Terminator
- B0015
- Terminator
- T9002
- Complete luxR+GFP system
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...