6/14/10 TWADDLE
In summary we have decided to pursue a mercury sequesting IGEM to be built from available parts, possibly. 1) Mercury transporter K205044 2) a glutathione part: possibly BBa_J61213 BBa_T20205 BBa_K289001 BBa_K289002 BBa_K289003... Our task is to evaluate the gluthione parts for one that we can couple to the mercury transporter:
6/21/10 TWADDLE ("da coach")
O.k. summary of our change of direction:
Since we have found some good examples of IGEMs that have been created to detect and/or sequester heavy metals we decided to shift gears and build a IGEM that can detect enteric bacteria: The idea is to build a device that can be use to sense bacteria in lake water. Lake Michigan beaches are susceptible to closure because of high E.coli counts. If we could develop an IGEM based upon the quorum sensing protein, autoinducer 2, the we could detect, indirectly the presence, even potentially the amount of bacteria in the water.
6/21/10 Joseph Hull(Joe)
How about we use the British Columbia 2009 stop light bacteria device by manipulating this device with a
AI-1 or AHL promoter to change this device to be sensitive to presence of AI-1
6/21/10 Chamberlin ("da geek")
Possible promoter LuxR is a constitutively expressed protein that can bind AHL. When bound to AHL it can stimulate transcription from the right hand lux promoter (pLuxR). In the natural system, this promoter controls transcription of the LuxI enzyme leading to a positive feedback loop that increases transcription from the right hand lux promoter. In addition controlling the transcription of luxI, the promoter also controls transcription of luciferase. 1 BBa_R1062Promoter, Standard (luxR and HSL regulated -- lux pR)
6/23/10 Cassidy
If we're going for the AHL aspect like Joe proposed what about checking into the utilization of...
Part:BBa_I729004 (DNA AVAILABLE/EXPERIENCE: NONE) Sensitive AHL Receiver This AHL receiver contains mutant LuxR.(I45F) It can express GFP more at AHL 10nM compared with wild type. (Ref. Collins, C. H., Arnold, F. H. & Leadbetter, J. R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55, 712–723 (2005))
or maybe...
Part:BBa_I729005 (DNA AVAILABLE/EXPERIENCE: WORKS!) AHL Reporter and Quencher GFP and aiiA are repressed Lux promoter GFP and aiiA induced by AHL. GFP will express if the amount of AHL exeed the capability of aiiA.
LB plates plus antibiotics - Joice P
"**Be sure agar is not to hot Cool to 55 degrees C. before starting procedure if agar is hot**"
1)Ampicillin- add 1 ml ampicillin (at 100 mg/ml) per liter of agar to obtain a final concentration of 100 ug/ml. Can mark with red line to signify amp.
2)Kanamycin- add 1ml kanamycin stock (at 50 mg/ml) per liter of agar to obtain a final concentration of 50 ug/ml. Can mark with green line to signify Kanamycin.
3) Tetracycline- add 1ml tetracycline stock (at 15 mg/ml)per liter of agar to obtain a final concentration of 15 ug/ml.Can mark with black line to signify tetracycline.
4)Chloramphenicol-add 1ml chloramphenicol stock (at 25 mg/ml)per liter of agar to obtain a final concentration of 100 ug/ml. Can mark plates with a single purple line to signify chloramphenicol.
--Joice2872 19:37, 30 June 2010 (UTC)
How to determine volumes to obtain a certain concentration Joice P
Use the following formula to determine amount of stock solution to use for any amount. Buy make sure units match!!
C1V1 =C2 V2 so C = concentration and V = volume
To convert mg/ml to ug/ml multiply by 1000
An example: stock solution of A at 100 mg/ml. You want 150 ml at 100 ug/ml. Use the C1V1 for what you want, and the C2V2 for what you have.
Just Plug n Chug. (100 ug/ml)(150ml) = (100000 ug/ml)(x)
15000=100000x
x = 0.15ml, or 150 ul (again remember you have to convert mg/ml to ug/ml, by multiplying by 1000)--
--Joice2872 19:38, 30 June 2010 (UTC)
6/28/10 Joseph Hull(Joe)
Preparing SOB-
SOB BROTH(DRY MEDIA) CatNo. S0221 Lot: S022130D0901
Scale: Mad by Adventurer Pro AV64
s/n: 8030391129 Error+ .67%
Calibrated by Cassidy
1) I used 4 - 250 mL capped elenmyer flasks
2) in two of them I added 6.97 grams of SOB media powder to 250 mL DI water
3)the mixed and dissolved on the hot plate
4) once dissolved, I separated the 2 - 250 mL SOB media into 4 - 250 mL elenmyer flasks and autocalved for one hour
Richard Lab:Electroporation of E. coli
This protocol is for the typical electro-transformation of E. coli done in the Richard Lab.
The consensus electroporation protocol should be consulted if deviating from the procedure outlined here.
Procedure
1. Chill the # electroporation cuvettes by floating them in an ice bath.
2. Remove # vials containing 100μl electro-competent cells from the -80°C freezer and thaw them with the iced cuvettes.
3. Prepare # micro-centrifuge tubes containing 900μl SOB media.
4. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes).
5. Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.
6. Place cells back on ice to ensure they remain cold.
7. Pippette 100μL of cell-DNA mixture to cuvette.
8. Wipe off excess moisture from outside of cuvette.
9. Place cuvette in chamber of electroporator.
10. Pulse the cells by pressing button on electroporator twice.
11. Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.
12. Let cells recover at room temperature for 30-60 minutes.
13. Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.
--Rchamberlin 19:59, 30 June 2010 (UTC)
Big Picture Revisited
So, am I correct in summarizing:
We are going to marry the BC stoplight/jammer device(s) with a device that is sensitive to AL2.
It may be possible to put this into E.coli, even though E.coli is making and sensitive to AL2: E.coli grown in dilute solution (plantonic form) should be green and as the concentration of bacteria, AL2, rises actually, then the color of the bacteria should shift from green to yellow to red. That of course is the ideal.
We could also build an AHL sensitive system but that will set us up to detect another type of bacteria....--Geotwaddle 20:11, 30 June 2010 (UTC)
New idea?
Can we merry stoplight idea with the AL2 sensor? New idea??--Joice2872 20:02, 7 July 2010 (UTC)
pGlo transformation successful
Our bacteria did take up the pglo plasmid after I placed 1 mL on LB amp/ara plate. We will now continue by transforming and cell banking more bacteria with the pglo plasmids while we await parts from the registry.
--Joice2872 18:53, 21 July 2010 (UTC)
Parts Needed and Found
We have identified 4 parts that we will need for the project.
Two parts are in the 2010 distributions:
Plate 2 - 20A
BBa_I729006
(pSB1A3)AHL reporter and aiia device
T9002
S01656
Express LuxR and aiiA constantly. AiiA degrades AHL as signaling molecule. Express GFP when the AHL concentration exceed the capacity of aiiA,.
Plate 1 - 1F
BBa_I13453
(pSB1A3)Pbad promoter
I13453
PBad promoter from I0500 without AraC.
Usage and Biology
Has been used as a second promoter in a system containing BBa_I0500 (PBad+AraC). In this system, it showed behavior qualitatively indistinguishable from the BBa_I0500 copy of PBad. Has not been tested independent of AraC. A second part, BBa_I13458, should allow decoupling of PBad and AraC.
See this OpenWetWare article on pBAD and lac promoters for additional usage and biology information
Characterization
For the 2009 iGEM competition, British_Columbia characterized BBa_I13453 in the context of a pBAD promoter family. For the results of this characterization, see here.
I have asked Professor T to order pBAD strong BBa_K206000 and pBAD weak BBa_K206001 that were submitted by British Columbia last year.
|
"
