Notebook

March

Brainstorming

Project ideas:
Pests
Bacterial Pest attractor (engineer bacteria to produce a substance, such as a pheromone or smell, that attracts and kills pests such as insects)
Natural pesticides (engineer bacteria to produce a substance that repels or kills pests, such as those that harm crops, that can later be implemented into plants)

Water/Environment:
Desalination of water (engineering bacteria to get rid of salt in salt water in order to make drinkable water from sea water)
Getting rid of estrogen mimicking compounds in water (engineering bacteria to degrade them or sequester them)
Waste treatment (engineering bacteria that can be integrated into waste water treatment)
CFCs (engineer bacteria to produce metabolites that break down chlorofluorocarbons—compounds that contribute to the degradation of the ozone layer)

Health
Stomach ulcers (creating a medication containing engineered bacteria to specifically target and kill Helicobacter pylori – the organism that causes stomach ulcers)
Diabetes (engineered bacteria as Islet cells to produce insulin)
Allergies/Immune system (engineer bacteria to produce antihistamines or alter epitopes)
Bone density (engineer bacteria to produce and secrete calcium and other compounds to help heal broken bones or to prevent osteoporosis)

Process Improvement
Oil fractionation catalyst (engineering bacteria to improve the separation of crude oil into valuable fraction and waste fraction)
Nitrate fixation (engineer bacteria to improve nitrogen fixation so not as much fertilizer is needed)

Kill Switches (engineering bacteria to undergo induced or programmed cell death in order to control the organism)

The chosen project

After dividing in to a few groups based on which project was preferred, each group made a presentation on the proposed project. In the end, we decided to go with the glucose sensing project.

Project outline

Possible things to consider

Glucose and insulin
-Detection of blood glucose levels (glucose-specific promoter?)
-Insulin production release (measure/regulation of production?)

Immune response
-Possibly use "bactoblood" strain from Berkeley 2007

Kill switch
-Control and regulation?

Delivery system
-Intravenous?
-NASA Biocapsule?

April

We divided the team into sub-groups. Each group assumed responsibility for a different aspect of the project.

Sub-groups:

-Wiki and Mascot Design

-Biosafety

-Human Practices

Wiki and Mascot Design:

This group is responsible to obtaining and uploading all necessary information to the wiki. This includes team pictures and biographies, experiments and results, and the projects of the other sub-groups. This will be a priority over mascot design.

Biosafety:

This group must demontrate the safety protocols that the Lethbridge team practices in the lab.

Human Practices:

This group is responsible for letting the poublic know what our team is and what we are doing. This can be done creatively. The point it to raise awareness for iGEM, synthetic biology and diabetes.

May

May 8, 2012: Tansformation of Parts from Kit Plate

-B0034 in pSB1A2

-K093005 in pSB1A2

-J23100 in J61002 (Amp)

-E1010 in pSB2K3

Overnight cultures were also made of:

-pMA-T K33109 (HST-1)

-pSB1AK3 B0014

May 9, 2012: Restriction of Parts

Master mix was prepared by mixing the ingredients together in order, as shown above. 20µL worth of master mix was combined with 5µL of plasmid DNA (1-5).

pMA-T K331009 (HST)

pSB1AK3 BBa_B0014

Incubated at 37˚C for 30mins then stored at -20˚C.

Colonies were also picked of :

-E1010 pSB2K3

-J23100 J61002 (Amp)

-K93005 pSB1A2

-B0034 pSB1A2

May 10,2012: Restriction of Parts

Master mix was prepared by mixing the ingredients together in order, as shown above. 20µL worth of master mix was combined with 5µL of plasmid DNA (1-7).

-J23100 (J61002)

-J23100 (J61002)

-B0034 (pSB1A3)

-B0034 (pSB1A3)

-E1010 (pSB2K3)

-E1010 (pSB2k3)

-K093008 (pSB1A2)

Incubated at 37˚C for 30min then stored in -20˚C.

May 22, 2012: PCR of J23100-Mlc

PCR Parameters:

Initial denaturation 95˚C 3mins 30 cycles of:

-denaturation 95˚C 30s

-annealing (various) 30s

-elongation 72˚C 1min

Final elongation 72˚C 7mins

PCR Samples:

PCR product expected size: 214 bps

1.5% agarose gel

Ran at 120v for 1 hr

May 23, 2012: Ligation of PCR products into pGEM Vector

1) prefix –J23100-mlc-spe cut site (Red tubes) (sample 3 from May 22nd PCR)

2) Prefix-(fusion)-E1010-suffix (Black tubes) (sample 1 from May 23rd PCR)

Contents:

-2x Rapid Ligation Buffer 5µL

-pGEM-Tor pGem-T Easy Vector 1µL

-PCR Product 3 µL

-T4 DNA Ligase 1µL

-Miliq H2O 1 µL

-Ligate at room temp for 1h 15 mins

• One of each sample ligated overnight @ 4˚c

• 2 of each ligated at room temperature for approx. 1hr

May 23, 2012 Transformation of J23100-Mlc and E1010 fusion

For each ligation mix:

-Added 2µL of ligation mix ( E1010 pcr + pGEM; J23100- mlc + pGEM) to 20.0µL of DH5α)

-Incubated on ice for 30mins

- 45secs in water bath @ 42˚C

- immediately incubated on ice for 5mins

-Added 400µL of SOC media; resuspended

-Put in shaker for 1hr @ 37 ˚C

-200µL cultured onto plate(4 plates, Amp)

-Incubate @ 37 ˚C for approximately 16hrs

June