Helpful Results/Links

FACS results
Plate Reader Data
Growth Curves
Plate Reader Protocol
Stephanie's Week 3 Powerpoint
Stephanie's Week 5 Powerpoint
Harvard iGEM
About Me

Week 1

Doing some reading on Fec now. Really brief notes and some of the more interesting readings follow.
Fec genes encode proteins essential for ferric citrate transport in e.coliK12.
FecA is an outer membrane protein
=N-proximal 79-residue extension: deletion of this extension abolishes induction by ferric citrate but retains feric citrate transport: Kim et al, Transcription induction of the ferric citrate transport genes via the N-terminus of the FecA outer membrane protein, the Ton system, and the electrochemical potential of the cytoplasmic membrane Kim article
Gene regulation by transmembrane signaling Some really nice info on structure of Fec and interactions with ferric citrate
Biocyc

All Medline abstracts: PubMed | HubMed

NanoDrop results (6/20) performed by Ellenor and Stephanie:
(1.5 uL used out of a 30uL elution with nuclease free water)

S: 10.9 ng/uL
S2: 15.4 ng/uL
B: 59.1 ng/uL
B1: 25.1 ng/uL

Brainstorming

Brainstorming for the two-component systems (really for my own use for now - not expected to be coherent)
Structural comparison of the PhoB and OmpR DNA binding/transactivation domains and the arrangement of PhoB molecules on the phosphate box
-NMR used to determine 3DE structure of PhoB DNA-binding/transactivation domain. Very similar to OmpR DNA-binding/transactivation domain, except for conformation of the long turn region of PhoB (interaction site for sigma subunit, rather than interaction with alpha subunit for OmpR)

Interdomain linkers of homologous response regulators determine their mechanism of action
Focuses on OmpR and PhoB and, as the title suggests, supports that phosphorylation of sites (particularly N-terminus of both proteins) improves affinity to bind DNA. Isolated C terminus of OmpR is insufficient to productively interact with RNA polymerase.
I've been told by some of the lab members that OmpR is an inner-membrane protein and therefore cannot be used for our assays. It seems that we'll have to find another protein ...

The phosphoryl transfer domain of UhpB interacts with the response regulator UhpA
UhpB = histidine kinase protein that controls production of sugar phosphate transporter UhpT
UhpA = response regulator; when phosphate is transferred from histidine to aspartate, ability of kinase to bind to target DNA sequences and to alter gene transcription is altered.
Major result: indication that phosphoryl, transfer-dimerization of UhpB participates in specific binding of UhpA, in control of autokinase activity, and dephosphorylation of P-UhpA
... So I found this paper on a search on PubMed for e coli outer membrane protein signaling dimerization. However, I've found that UhpA resides in the cytoplasm, whereas UhpB is an inner-membrane protein. Boo.
More thoughts: we could potentially try to target some of these inner-membrane proteins to the outer-membrane. I don't know if this is really feasible - while we can attach the appropriate signal sequence, I'm not sure the environment would allow for correct conformation and activity of said proteins.

Week 2

6/27

Yesterday night I left some vectors & plasmids to ligate in the 37C incubator. Kevin took the vectors for nucleotide removal, and I speed Vac'd the plasmids. Originally I was going to make a gel and run the plasmids on multiple lanes for gel extraction, but given the large Nanodrop values (242 - 371 ng/uL) and the large amount of Midiprep used (50uL), Bill suggested that I PCR purify them instead, which gives 95% recovery rather than 80% recovery. A gel will still need to be run to confirm that the samples were indeed ligated.
The plasmids are Lpp+OmpA1+pet29 and Lpp+OmpA2+pet29.

Reconstitution into 30uL of H2O per plasmid (two plasmids total - OmpA1, OmpA2),
split into two samples each (so 15uL total volume)
PCR purified (PCR purification kit from Qiagen)
Eluted with 50uL nuclease-free H2O per tube
prepared a gel using TBE
prepared 5 lanes

Lane 1: 10uL ladder (1kB +)
Lane 2: 1uL OmpA1 plasmid (diluted to 20uL total using H2O)
Lane 3: 3uL OmpA1 plasmid (diluted to 20)
Lane 4: 1uL OmpA2 plasmid (diluted to 20)
Lane 5: 3uL OmpA2 plasmid (diluted to 20)

The gel was run at 130V for 45 minutes.
4 tubes total should remain: 2 tubes of each type of plasmid. 2 tubes should have 49uL left (labeled "1"), 2 tubes should have 47uL left (labeled "2"). After gel ran for 45 minutes, bands appeared about 3/4 way down the gel. Ethidium bromide (2uL) and 100mL TBE buffer used, put on shaker for 45 minutes. Visualized under Trans-UV.

Nanodrop, 6/27:
10mer: 122.3 ng/uL
15mer: 156.3 ng/uL
20mer: 167.4 ng/uL
plasmidOmpA1: 39.6 ng/uL
plasmidOmpA2: 42.8 ng/uL

We Speed Vac'd the plasmids and resuspended both in 33uL of nuclease-free H2O. To calculate volumes of sample needed , we approximated that the vector was 5800 in size and the insert was 50. Therefore, we'd need 1200ng of vector and 103ng of insert.
Protocol for Ligation of Plasmid given Nanodrop results and volumes available
OmpA1 or A2: 10uL
10mer, 15mer, or 20mer: 1uL
buffer: 1.5uL
ligase: 1uL
dH2O: 1.5uL
(total of 15uL)
Leave in PCR machine (left in the one at George's workstation) overnight for 15C. The extra 10,15,and20mer tubes were put in the "Mike Strong iGEM plasmid" box in the freezer in the small laboratory. We used shorthand for the small PCR tubes: 110 means OmpA1+10mer, 115 means OmpA1+15mer, 210 means OmpA2+10mer, etc.

6/28

George, Perry, and I transformed some of the BioBricks into 20uL of Top Ten cells.
We want to change the pL promoter because it does not allow for constitutive expression. Some of the Biobrick parts we're using may facilitate this.
Also: grew cells in liquid media, digested BB, looked at sequences and discovered that the BB isn't what it should be - should be about 800bp, with ribosome binding region and terminator region, but was only about 300bp, with terminator and something following it.

6/29

... oh lord. i have so much more to add. sorry for the delay guys ... mad busy day.

George, Perry, and I arrived at 8:15am to start to prepare the bacteria for the FACS appointment at 3:30. We took the optical densities of the cells we grew in liquid medium overnight and got 1.785, 1.793, 1.780.

9am: We prepared 3 tubes to 2mL each, with dilutions of 1:40, 1:80, 1:100. 2uL of Amp was added in each, and the following:
1:40 - 50uL cells, 1498uL LB
1:80 - 25uL cells, 1748uL LB
1:100- 20uL cells, 1792uL LB

9:15am: Made and set a 1% agarose gel for the digest we prepared yesterday.
Also helped Perry with some of his colony-PCRs. Gel has 3 lanes- 1: ladder, 2: yesterday's digest, 3: Perry's sample. Will be visualized using SyberGold

10:30am: OD of 4-hr samples. Realized we had not diluted them enough. Diluted 1:100 for the spectrometer (so we did not use up too many samples), had reading of 0.03 for 1:40, 0.018 for 1:80, 0.013 for 1:100. Decided to re-dilute 1:100 for samples originally marked 1:40 and 1:100, and 1:30 for sample originally 1:80. Gives us an absolute OD of 0.03 for the 1:40, 0.01 for the 1:100, and 0.05 for the 1:80. (made 2mL total for each sample, adding in cells, LB, and Amp)
1:40- 20uL cells, 2mL LB, 2uL Amp, 1:80 - 65uL cells, 2mL LB, 2uL Amp, 1:100 - 20uL cells, 2mL LB, 2uL Amp.
Also: prepared 2hr samples

11am: George prepares HSL solution using 200proof ethanol.

11:30am: Prepare the 1-hr incubations

12 noon: OD of 4-hour induction, right before protein added. 1:100 dilution done for spectrometer. (10uL cells, 990LB). The numbers below are the raw numbers from the machine - so our samples actually have OD 100x this.
1:40 - 0.002
1:80 - 0.002
1:100 - 0.001
Since we didn't have as much volume of cells as we had anticipated, instead of doing 2mL cell samples as planned, we used 1mL of cells with 10.5 uL HSL (diluted by George - see his personal notebook page). 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, with 1nM HSL.

1:30pm: Induced the two-hour samples. First, OD taken of 2-hr and 1-hr (1hr had been growing for about 1.5 hours at this point).
Again, all ODs diluted 1:100 first.
Two hours: 1:40 - 0.078, 1:80 - 0.035, 1:100 - 0.005. One hour: 1:40 - 0.016, 1:80 - 0.008, 1:100 - 0.007. Also, we had left the 4-hour cells on the bench (from before) - ODs of 0.002 (1:80) and 0.004 (1:100).

2 samples from the 2-hour 1:100 (0.005 OD) were used to make the 10, 100nM HSL.
Since the ODs were already so high for the 1-hr, we also decided to take our original overnight liquid cultures and dilute them for the 1-hr. We used the same formula as above and diluted to 1:80 and 1:100.

2:30pm: 1-hr rediluted samples gave 0.003OD (1:80) and 0.002 (1:100). We used the 1:80 for the 1nM HSL and the 1:100 for the 10, 100nM HSL.
A negative control was also created from 1mL of the 1:80 solution.

3:15pm: All samples spun down for 2 minutes at 10,000rpm, supernatant removed, reconstituted with 0.5mL PBS (1x)

Colony counts from 06/28:

275uL
OmpA1 + 10mer: 167
OmpA1 + 15mer: 266
OmpA1 + 20mer: 224
OmpA2 + 10mer: 60
OmpA2 + 15mer: 9
OmpA2 + 20mer: 28
10uL
OmpA1 + 10mer: 15
OmpA1 + 15mer: 8
OmpA1 + 20mer: 0
OmpA2 + 10mer: 1
OmpA2 + 15mer: 0
OmpA2 + 20mer: 0

6/30

Helped miniprep some Biobricks, but 3 of the labels smeared and I was unable to discern them. PCR'd them, Will run an E-gel.
E-gel:
Lane 1: 1kb+ ladder
Lane 2: Sample 1
Lane 3: Sample 2
Lane 4: Sample 3
Rest of lanes loaded with water as instructions dictate.

7/01

Did another miniprep of some Biobricks that Perry had grown in liquid culture. The tubes are in the freezer at my workstation. Perry helped me visualize the Egel from yesterday (the machine is somewhat broken ... I had left the gel in the fridge overnight. Though some of the gel diffused, the bands are still distinguishable). The samples were known to be either R0051, B0015, or T9002. Band 3 ran the slowest, indicating the highest molecular weight, and therefore T9002. B0015 is 129 bp whereas R0011 is 55bp, so sample 2 (which ran the furthest) was likely to be R0011, and sample 1 was B0015.

Readings and Brainstorming

Idea: increasing ligation efficiency
According to a paper I just read, (Lund et al) "temperature cycle ligations" may provide a 4-8 fold increase on cloning efficiency, since the cycling balances high enzyme activity and DNA annealing. The temperature cycle should run for 12-16 hours, cycling between 30 second bouts at 10C and 30C.

Readings toward QS and lux
Bacterial Transformation Experiment James Slock has a detailed protocol for transforming the lux genes into e.coli. Actually, the transformation protocol is the same as what we've been doing, but there is some specific info on lux stuff. Not really essential, I guess.
MIT Parts Registry: Overview of LuxR system
[ww.ai.mit.edu/projects/ cellular-robotics/rweiss-dna6.ps Paper] Really nice, but needs to be converted to pdf - my Mac did this automatically ...
MIT Parts, BBa_F2620
spatiotemporal control reading Look at the supplementary information as well.
Paper ...some other genes that may be turned on by HSL are mentioned ...
somewhat relevant ... but not completely Seems that the e. coli were grown with OHL... and a different strain was used
BL21 More info on the cells we're using

look in methods for treatment of HSL powder ... mammalian cells, but for method only
shlo/notebook/luxfluorescenceduration

Week 3

07/02

Nanodropped some of the samples from yesterday and Saturday before Perry digested them and we sent them out for sequencing.

Sample	Nanodrop result (ng/uL)
S03623, Sample 1	104.5
S03623, Sample 2	111.2
S03608, Sample 1	126.7
S03608, Sample 2	110.3
J37034, Sample 1	196.5
J37034, Sample 2	97.6
J23039	65.8
R0011	61.4
R0140	86.1
T9002	185.1
B0015	71.6
R0051	100.2

We wanted to construct a growth curve for our cells, so we took the refrigerated BL21+I13263 cells (from Friday) and made 800uL of different dilutions for the plate reader. Ampicillin and LB were added to each sample, and samples were made for 1:10 (wells A1-3), 1:20 (B1-3), 1:30 (C1-3), 1:40 (D1-3), 1:50 (E1-3), 1:60 (F1-3), 1:70 (G1-3), 1:80 (H1-3), 1:90 (A4-6), 1:100 (B4-6), and of the BL21 cells (1:10, C4).
Also, diluted the OHHL to 0.05M. There is 938uL total.

Grew liquid cultures of I5311, OmpA1+his, I13522, T9002, and I13263. These will be used when we induce tomorrow and use the Plate Reader to detect fluorescence.

See Perry's notebook for more info on this.

07/03

shlo/notebook/growthcurves0703 ... Growth curves for BL-21 with the lux insertions. Most of them look pretty good; should serve as a general reference for all groups, until (if?) you make your own growth curves.

Helped Perry with some ligations/plating, and started a 5-hour fluorescent/induction experiment. First, I loaded the machine with cells (dilution - 1:10 ... maybe will dilute in greater amounts next time, eg 1:100?) and watched OD. Around 0.3OD I took them out, transferred to a clear/black 96-well plate (next time, will start with one of these), and induced cells with OHHL.

Wells A1-3: I5311 (constitutive YFP, kan)
Wells A4-6: I13522 (constitutive GFP, amp)
Wells B1-3: BL-21 with PET29 OmpA1+His (negative control, kan)
Wells B4-6: BL-21 with I13263 (negative control, inducible for YFP, amp)
Wells C1-3: BL-21 with I13263 (10nM OHHL added)
Wells C4-6: Top10 with T9002 (10nM OHHL added)
Wells D1-3: BL-21 with I13263 (100nM OHHL added)
Wells D4-6: Top10 with T9002 (100nM OHHL added)

Also, digested some E0240 and plated S03608 and S03623, along with B0015. We'll use this to re-create the J37034 that hasn't seemed right (though our own constructs will have different promoters ... which is good in the long run). All of these samples (plates and microcentrifuge tube) are in the incubator, and someone will have to move the plates to the fridge and the tube to the freezer tomorrow.

07/04

Took plates out of incubator, put into fridge
F2620, top 10: lawns of colonies
JL0159: lawns
S03623-B0015: a few
JL0159-PDZ1+2: minimal growth
JL0159-PDZ2: some growth
S03608-B0015 in Top 10... are these air bubbles or colonies?
JL0159-PDZ1: Minimal growth

I put the E0240 in the freezer. Be careful; the non-digested microcentrifuge tube is there as well!! The digested one is clearly labeled "digested".
shlo/notebook/0704fluorescence

07/05

Prepared a colony PCR using some of the plates that I moved yesterday.
The PCR looks really bad/has hardly any bands, which is really confusing. We might have to redo this all ...

07/06

Helped Perry prepare a colony PCR of F2620 and a few other samples
Planning to do some transformations at the end of the day, including some of the parts we found: J06702, F2622, F2621, I15030 (we should have this somewhere), !6042, I13273, J37015 (?)

Found Imperial's page from last year, with testing of the T9002 part we're using. Interestingly, as they varied AHL concentration, fluorescence did not seem to change too significantly. Imperial T9002 Results

Additional Readings and Thoughts

Trying to read to trigger brainstorming ideas for fluorescence assay as well as gauge what to expect.

Harris poses a good question: why do we have luxR/luxI constructs in different bacteria? Will probably be a lot less sensitive than if we had both in one ... though perhaps the applications of having a sender/receiver are more diverse? Time to discuss..

Again, the Andersen et al paper (gfp-based N-acyl homoserine lactone sensor systems for detection ...) is extremely helpful in this regard:

Their construct (e. coli MT102) was much more sensitive to OHHL relative to other proteins in the acyl-homoserine lactone family.
1 nM OHHL concentrations did not cause fluorescence to be any higher than background. Significant fluorescence began at about 3nM OHHL. 100nM OHHL was only slightly higher in fluorescence compared to 10nM OHHL; as I've noted before, "maximal induction" was coined to be at 10nM OHHL.
GFP: excitation of 475nm and emission at 515nm
Interestingly, the fluorescence peaked around 250 minutes, and then started to decrease (... this could do with their different GFP constructs, though).

Ripp et al: Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

We've been talking about trying to measure amounts of OHHL produced, so we can detect whether our lux I construct is working. n this paper, OHHL concentrations were analytically determined using liquid chromatograph-mass spec. At least it's feasible, though I don't think our lab could/would do this.
Induced E. coli OHHLux bioreporter using synthetic OHHL.
- "significant" signals were considered to be those 2 standard deviations above background.
- Saturation-type behavior was observed at OHHL concentrations exceeding 50uL ... which is much higher than anything we've used
- Found a linear-type response at OHHL concentrations from 20nM to 2uM

Burmolle M et al: Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry

To figure out dose-response, OHL and samples shook at 37C for 20 hours (including antibiotic and LB, 5mL total). Afterward, 1mL was washed once in PBS (for each sample) and resuspended in 3mL PBS for fluorescence readings

A bunch of papers also seem to reference TLC (thin layer chromo) as a possibility to detect/characterize OHHL levels.

Week 4

7/9

Perry, George, and I did a number of colony PCRs on various samples. Results below. The ones that worked are marked.

Lane 1: Ladder
2: J37015, 1
3: J37015, 2
4: J06702, 1 (as expected)
5: J06702, 2
6: I6042, 1 (as expected)
7: I6042, 2 (as expected)
8: I13273, 1
9: I13273, 2 (as expected)
10: F2622, 1 (as expected)
11: F2622, 2
12: H2O

Lane 1: Ladder
2: F26-I07
3: S08-E40
4: S23-I07 (as expected)
5: S23-E0240 (as expected)
6: F26-E40
7: S08-I07 (as expected)
8: F2621, 1 (as expected)
9: F2621, 2 (as expected)
10: I15030, 1
11: I15030, 2
12: H2O

Made liquid cultures of a number of samples for plate reader tomorrow.
Also, left one portion of T9002 incubating with about 1000 nM OHHL to serve as comparison/control of fluorescence, to J37015.

7/10

Plate Reader

Prepared plate reader for fluorescence with OHHL.
A1-3: T9002 (-), 10nM OHHL
A4-6: T9002 (+), 10nM
B1-3: T9002 (-), 100nM
B4-6: T9002 (+), 100nM
C1-3: I13273, 10nM
C4-6: I13263, 10nM
D1-3: I13273, 100nM
D4-6: I13263, 100nM
E1: B0015, negative control
E2: T9002(-), no induction
E3: T9002(+), no induction
E4: I13273, no induction
E5: I13263, no induction
E6: I13522, no induction (constitutive GFP => positive control)
Note: T9002 (+) indicates that the T9002 sample was incubated with 1000nM OHHL.
The samples were all put in 1:20 (sample:total volume), total volume = 200uL, inclubes LB + Amp.
Induction expected to occur around 1hr20min after dilution, at 0.3OD, around 12:15.

Readings: SdiA and AHL

Interestingly, on our plate reader today, T9002(+) samples grew consistently faster than the T9002 (-) and other samples. The I63 and I73 samples grew at the same rate despite being in different types of cells (Top 10 vs BL21).
Some papers seem to suggest that SdiA acts similarly to a LuxR and is involved in cell division processes (though papers also mention that exact mechanisms remain unknown). One paper has found that addition of AHL to e coli should not affect their overall growth, though a number of genes are upregulated and downregulated, including OmpC (upregulated). [[1]]

Additional Readings:

Colony PCRs

Prepared 3 different colony PCRs & E-gels.
Extension for 1min15sec

Lanes 2-6: F2620
Lanes 7-12: B0015
Extension for 2min15sec

Ladder
I15030 1
I15030 2
F26-E 1
F26-E 2
F26-E 3
F26-I13507 1
F26-I13507 2
F26-I13507 3
S08-E0240 1
S08-E0240 2
S08-E0240 3

Extension for 3min15sec

Ladder
J37015 1
J37015 2
J37015 3
J37015 4
J37015 5
J39-T9002 1
J-T 2
J-T 3
J-T 4
J-T 5
J-T 6

7/11

Note: We had set up some fluorescent readings on the plate reader to run for a few hours yesterday, but somehow the PC restarted and failed to save/restore our information. Other than a few observations that we had made during the process - such as T9002 (+) growing much faster than T9002 (-), all our data was lost ... very, very unfortunate and aggrevating.

Plans

Figure out J-T: start with low concentration, see if fluorescence spikes (which might indicate quorum-like activity) rather than slow growth of fluorescence.
Re-Colony PCR some of the constructs
- I5311 (... actually, we've never PCR'd this)
- F2620-I13507
- S08-E40
- F2620-E40
- I15030
New growth curves?

Setup for FACS

ODs and fluorescence taken from the plate reader to figure out dilution and efficacy of some of the overnight dilutions/combinations.

Induction, 100nM final concentration OHHL

Sample	OD	Fluorescence (excitation485, em38)	OD after one hour of growth, at induction
T9002 (+)	1.486	675.85	not used
T9002 (-)	1.212	212.61	0.379
I13273	1.283	208.69	0.430
F2620-E0240	1.487	191.98	0.527
J23039+T9002	1.174	4698.8	0.317
B0015	1.470	174	0.496
I13522	1.571	7978.7	0.397
I5311	1.643	2258	0.482
S08-I07 w/T9002	1.313	2538	0.427
S08-I07 w/ I73	1.248	1701	0.395

Interestingly, the T9002 (+) had relatively little fluorescence compared to the constitutive samples. Perhaps the fluorescence had started to decrease over time, or perhaps the T9002 was not activated that strongly ... this is something we can measure over time with the plate reader ... if it does not turn off again and lose our data.

8mL LB + Amp (except for I5311, with Kan) added to enough uL of cells to make a "cell mass" of 371.5.

Results from FACS

All inductions were at 2 hours with 100nM OHHL.
We've also learned that we should do about a 1:50 dilution of the overnight cultures. 0.03 OD is probably optimal.

T9002 (non-induced) had some leaky fluorescence.
T9002 (induced) showed strong fluorescence as expected.
I73 (non-induced) had a little less leaky fluorescence than T9002, but still had some leakiness.
I73 (induced) showed strong fluorescence as expected. S08-I07 + T9002 had little fluorescence in the GFP but had lots of fluorescence for RFP.
S08-I07 + I73 had strong fluorescence for both YFP and RFP. It should be noted, though, that the count of YFP was around 500 at peak, while some of the induced were around 1250 at peak. Perhaps this has to do with inconsistent cell numbers?
S08 I07-T9002 had very little GFP (low count, both negative and positive) but strong RFP.
J39-T9002 showed strong GFP fluorescence.
15311 showed strong YFP fluorescence as expected, with peak counts around 1600.
I13522 showed strong GFP, as expected, with peak around 1750.
B0015 served as the negative control
F2620/E40 (induced) showed no fluorescence, nor did the F2620/E40 (non-induced). Probably time to let go of the F2620.

Setup for Overnight Plate Reader

A1-3: T9002 (noninduced)
A4-6: T9002 (+100nM OHHL)
B1-3: I13273 (noninduced)
B4-6: I13273 (+100nM OHHL)
C1-3: F2620-E0240 (noninduced)
C4-6: F2620-E0240 (+100nM OHHL)
D1-3: J39-T9002
D4-6: B0015
E1-3: I13522
E4-6: I5211
F1-3: S08-I07 w/T9002
F4-6: S08-I07 1/ I73
G1-6: Swabs of J-T (noninduced)
1:100 initial dilution
shlo/notebook/flu0711
The decrease in fluorescence that is seen on many of the samples is probably due to the wells drying out over time.

7/12

I miniprepped some liquid cultures that were grown last night:

R0040
R0011
P0140(-1)
P0340(-1)
R0051

George and I also Colony PCR'd the biobricks that I listed in my plans for yesterday.

Ladder
I15030 1 (NO)
I15030 2 (NO)
I5311 1 (a little too big)
I5311 2 (a little too big)
I5311 3 (no band)
S08-E40 1 (as expected)
S08-E40 2 (as expected)
F20-I07 1 (as expected)
F20-I07 2 (as expected)
F20-E40 1 (as expected)
F20-E40 2 (as expected)

We will grow overnight cultures of:

JT
S08-E
S23-E
S08-I07
S23-I07
F26-E

Looks like the plate reader can do more than one wavelength, so having the RFP constructs is going to be really awesome.

Overnight Plate Reader

Hopefully, this will allow us to compare the shape of the J+T (full construct) curve versus the T9002+OHHL curve. We hope the T9002+OHHL will prove to be a somewhat linear growth in fluorescence while J+T proves to be sigmoidal (showing quorum activity).
Initial ODs were all about 0.05 or 0.06.

All of the following were swabs (touch of a pipet tip to the liquid culture, such that the initial number of cells is very low). Unfortunately, J-T was not available in liquid culture, so a direct touch from the plate had to be done.
A1-6: J-T
B1-3: I13522
B4-6: I13263
C1-3: B0015
C4-6: I13263 w/ 10nM OHHL
D1-3: I5311
D4-6: I13263 w/ 100nM OHHL
E1-3: I13273
E4-6: T9002
F1-3: I13273 w/ 10nM OHHL
F4-6: T9002 w/ 10nM OHHL
G1-3: I13273 w/ 100nM OHHL
G4-6: T9002 w/ 100nM OHHL
H1-3: J-T 1/ 10nM OHHL
Some Results: ShLo/notebook/0712overnightplate

7/13

Created a 96-well plate for the plate reader (overnight, will pick up tomorrow). read is on fluorescence for both RFP and GFP. I was more careful to do dilutions of cells rather than swabs ... so hopefully growth and number of cells will be more consistent. Final dilution for most of the samples ended up being .5uM of the original sample, though I varied this in some of the wells. Some things that are being tested:

Fluorescence of J-T with OHHL versus fluorescence of just JT versus fluorescence of T02 with OHHL.
Different relative concentrations of lux I (S08-I07 and S23-I07) constructs versus lux R (FE ... this part hasn't worked in the past so I can only hope). I probably should have used T02 but I didn't grow this overnight, and had to use a liquid culture that was about a week old (from refrigerator)...

Unless otherwise specified, the dilutions below are 1:100, add 1uL to final 200uL well for a 5x10^-5 final dilution. All are in LB+Amp.

'	1to3	4to6	7to9	10to12
A	J-T	J-T w/OHHL	FE w/OHHL	FE alone
B	S08-I	S08-I w/FE	S08-I w/T02	T02 alone
C	S23-I	S23-I w/FE	S23-I w/T02	S23 alone
D	T02 w/OHHL	I13522, 0.5 uL of original	I13522 w/ OHHL	I13522
E	2xS08-I w/ FE	4xS08-I w/ FE	S08-I w/ 2xFE	S08-I w/ 4xFE
F	2x S23-I w/FE	4x S23-I w/FE	S23-I w/ 2x FE	S23-I w/ 4xFE
G	10xS08-I w/FE	100xS08-I w/FE	S23-I w/ 10x FE	S08-I w/ 100x FE
H	T02 w/ 10nM OHHL	FE w/ 10nM OHHL	JT w/ 10nM OHHL	I13522, 1uL from original

7/14

Picked up plate from yesterday. RFP didn't seem to be detected at all, but I double checked my em/ex wavelengths online and they look right. Maybe I'll change them up a little and see if it helps.
Set up an OD plate to accompany results. Will probably come in tomorrow or late tonight to start new plate reader, etc.

Plate Reader

'	1to3	4to6
A	J-T	J-T w/100nM OHHL
B	S08-I	13522
C	S23-I	T02
D	J-T	JT w/ 100 nM OHHL
E	S08-I07	13522
F	S23-I07	T02
G	FE 1:100	FE 1uL

A-C rows: Original liquid overnight, 1:100 dilution, add 1uL of dilution to 200uL well.
D-F rows: 1uL of original overnight added to 200uL well for final 1:200 dilution.

Grew up some liquid cultures of BL-21 to start testing those in similar ways:

I13522
J-T
S08-I07
S23-I07
T02
I73
S08-B
S23-B

All of these constructs were colony-PCR'd by George and Perry, though the gel did not appear on either of their sites so I'm posting it here. (I found it in the quorum folder on the usual imaging computer) They deserve full credit, however, and should feel free to take this off and replace with a link to their page instead.

Well 2: J-T
Well 3: S23-I07
Well 4: I11
Well 5: I22
Well 6: S08-I07
Well 7: S08-B1
Well 8: S08-B2
Well 9: S23-B1
Well 10: S23-B2
Well 11: T02
Well 12: I73

It's unclear how long the BL-21s will take to grow, so I stuck them in the shaking incubator near the plate reader at 25C (since I suspect they'll take a shorter time to saturate), and will pick them up either late tonight, or more likely, early tomorrow morning to start another plate reader.

Started a colony PCR on the P-R (tetR-creating) constructs. They are in the PCR machine closest to the door. The plate (with streaks) is in the incubator in the other room, near the UV machine.

Will redo the promoters from scratch, adding a new one for more options, with phosphotase treatment before clonewell this time. Also, will make enough miniprep to send some for sequencing and use the rest for digest/phosphotase etc.
Grew up in liquid culture:

R0051
R0011
R0052 (kan)

To do on Sunday (tomorrow): Miniprep (at least); start another plate reader ... maybe use BL-21 constructs this time?

7/15

Miniprepped the promoters: R0011, R0051, R0052
Elution in 50uL
Though ODs were pretty low for R0051 and R0011 (42.9 and 45.6, respectively), I still took all samples and removed 10uL for sequencing.
9:50am, put into incubator: Digested 38uL of each sample with SP. They are in the plate incubator in the other room. Should be ready by noon today. Remember to phosphotase-treat before clonewell this time ...
Also, set up another plate reader with the BL-21, am testing for 'quorum like activity' versus induction in J-T and T constructs, and also interaction between S08-I07 and S23-I07 with the T. Added a positive RFP control this time, though I took fluorescent readings of the saturated cultures and I'm a little concerned because of the difference (already!) between the S-I and positive control in terms of RFP fluorescence
OD and Fluorescence readings of RFP Saturated Liquid Cultures:

J04450 (constitutive): 1.695, 4987.1
S23I07: 1.807, 752.85
S08I07: 1.690, 512.57

Overnight Plate Reader Setup

All samples used 1uL of original (saturated) liquid culture into the 200uL well.

A1-3: J-T alone
A4-6: J-T with 100nM OHHL
B1-3: T02 alone
B4-6: T02 with 100nM OHHL
C1-3: T02 with S23I07
C4-6: T02 with S08I07
D1-3: J04450
D4-6: I13522

Week 5

7/16

Came in early to colony-PCR some of the promoter/tet constructs. Most of the plates had appropriate growth, with the exception of R0052+P0140, which had hardly any (3?) colonies.

Ladder
R52-P01-1(worked)
R52-P01-2
R51-P01-1(worked)
R51-P01-2(worked)
R51-P03-1(faint)
R51-P03-2
R11-P03-1
R11-P03-2(worked)
R52-P03-1(worked)
R52-P03-2
R11-P01-1

Plate Reader

'	1to3	4to6	7to9	10to12
A	J04450	I13522	I13263	S23-I / 12 has just LB
B	J-T	J-T + 100nM OHHL	J-T + 10nM OHHL	S08-I07
C	T02 noninduced	T02+100nM OHHL	T02 + 10nM OHHL	S23-E
D	0.5uL T02 + 0.5uL S23-I07	0.25uL T02 + 0.75uL S23-I07	0.75uL T02+ 0.25 uL S23-I07	S08-E
E	0.5uL T02 + 0.5uL S08-I07	0.25uL T02 + 0.75uL S08-I07	0.75uL T02+ 0.25 uL S08-I07	T02

The "0.25uL" samples were actually diluted down, such that the addition of cells was equal to 0.25uL of the original liquid overnight culture, but the volume was within our pipet's capabilities.
Read at the following wavelengths:
584/607 (RFP)
485/538 (GFP)
Two wavelength sets, one at upper limit and one at lower limit of plate reader capability, to try to detect cell number through scattering.

7/17

Learned how to use the microscope (guess this is pretty important, since now I'm developing new assays with this), will do an overnight plate (per usual - I'm getting pretty efficient at these, at least), will grow up some liquid cultures. Also, troubleshooting/randomly trying a new diluting/quorum(?) assay with the JTs.

Plate Reader Results from last night:

Reading notes:
Recombinant GFP E coli: [3]

"Time-dependent changes in bacterial growth were monitored by measuring OD595"
Macromolecules present in LB media such as sugar and carbohydrates absorb at A400-600

Problems with Plate Reader Assay

LB dries out before mid-log phase is achieved
- Perhaps dilute such that initial cell number is higher: use 1:50 or 1:100 rather than 1:200?
- Use 300uL in each well instead of 200
Control (13522) shows increasing fluorescence (divided by) OD over time
- May be function of cells not yet reaching log - protein expression differs over growth curve?

Next FACS Appt Ideas

Bring samples at different points of growth (J-T). Take OD before going, measure fluorescence. Determine whether cells at beginning of growth are already fluorescing (residual fluorescence? self-induction?)
- Cells would have to be collected throughout the day, stored at room/non-permissive temp. Also, shake: so that cells do not settle and therefore cause false quorum reading?
- How long do cells last in PBS?

Misc

Continue to use microscope to look at individual cells, determine when fluorescence turns off and whether self-induction actually occrs
To determine quorum-activity:
- I13263 ... assuming luxpL is downregulated at a concentration of dimerized luxR/OHHL that is above quorum concentration, fluorescence should shoot up at quorum but then be downregulated, such that the liquid culture does not fluoresce.
- The "slope" of fluorescence at supposed quorum can be compared to induced ... not sure if this will imply quorum occurs or not ...

Set up an OD plate reading for tonight, with higher concentrations of cells to begin with, the plate cover on, and 300uL of media.
Grew up 9 different liquid cultures (mainly BL-21, maybe except I13263)

Some exciting microscope things that I've done ... to come soon. Yay images!
File:Image1slo0717micro.jpg
To Do tomorrow:

Fluorescence assays with 13263, lower dilutions
More controlled scope pics with regular time points, this time with endpoint fl/OD readings
Make more lysine-plate covers
Clean up this wiki. Word.

Shlo/notebook

Week 1

Brainstorming

Week 2

6/27

6/28

6/29

6/30

7/01

Readings and Brainstorming

Week 3

07/02

07/03

07/04

07/05

07/06

Additional Readings and Thoughts

Week 4

7/9

7/10

Plate Reader

Readings: SdiA and AHL

Colony PCRs

7/11

Plans

Setup for FACS

Results from FACS

Setup for Overnight Plate Reader

7/12

Overnight Plate Reader

7/13

7/14

Plate Reader

7/15

Overnight Plate Reader Setup

Week 5

7/16

Plate Reader

More Random Readings

7/17

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