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From 2010.igem.org

Contents 1 6/14/10 TWADDLE 2 6/21/10 TWADDLE ("da coach") 3 6/21/10 Joseph Hull(Joe) 4 6/21/10 Chamberlin ("da geek") 5 6/23/10 Cassidy 6 LB plates plus antibiotics - Joice P 7 How to determine volumes to obtain a certain concentration Joice P 8 6/28/10 Joseph Hull(Joe)

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.

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 19:09, 30 June 2010 (UTC)

6/28/10 Joseph Hull(Joe)

Preparing SOB- SOB BROTH(DRY MEDIA) CatNo. S0221 Lot: S022130D0901 Scale 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