Part 0: Introduction

Part 0: Introduction

Last Meeting Summary

- The needed parts have been requested, Randy has said he’ll ship on Nov 11^th. Enzymes and buffers have been obtained.

- The temperature sensor project name is tentatively the “Cell-see-us” Thermometer, thank Andrew for that one :)

- It’s been agreed that both the temperature sensor and etch-a-sketch will be assembled and tested. Note that both can be assembled in a total of 10 addition operations.

- It’s been generally decided that the undergrads (Hannah/Emanuel/Joseph) will be working together at Steve’s lab (MSB4232) to combine all the parts. Please let us know if you want in for scheduling purposes…

To Do

- Assemble, transform E. coli, do multiple trials. Make video (I’m hoping there’s a camcorder available?).

- I can make the presentation, everyone please email me any materials/thoughts/suggestions you think may be helpful.

Part 1: Overview of Cell-See-Us Thermometer and Bacterial Etch-a-Sketch

Tuneable Temperature Sensor v2 (using LacI ts + other parts from Registry 7.05) [BBa_J11021]

http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6155

pBad/araC
I0500

B0034

lacI ts
J06501

B0010

B0012

LacI
R0010

B0031

C0056

B0010

B0012

Prm +
I12006

B0032

E0040

B0010

B0012

LacI
R0010

B0031

mRFP1
E1010

B0010

B0012

Explanation Summary:

Keeping the cells in a L-arabinose solution induces the cells to produce a specific steady state value of lacI monomers. The portion of these monomers that is in dimer form decreases with temperature. As temperature increases, the LacI dimer concentration decreases, resulting in reduced inhibition of the R0010 promoters. Hence RFP concentration increases. At the same time, more cI is produced, which inhibits Prm +, resulting in less GFP at steady state.

Explanation Details:

1) Consider a system with just the “LacI” (pLac) promoter and associated coding regions. The cells are in an IPTG solution, which results in the promoters being induced, resulting in 434 cI and RFP being produced at a “maximal” rate.

2) Now consider some LacI ts added to the system, which forms a dimer at some equilibrium concentration. At [LacI dimer] = 0, 434 cI and RFP are produced at maximal rates. At some concentration of LacI dimer, the transcription rate becomes “minimal” since LacI inhibits the “LacI” promoter (pLac). Between the maximal and minimal rates, there is a linear region where transcription rate gradually decreases as a function of LacI concentration. (see Graph 1)

3) The pBad promoter can be induced by keeping the cells in a solution of L-arabinose. When induced, lacI ts is produced. The concentration of L-arabinose can be set so that at 37˚ the [LacI dimer] concentration is centered in the linear region. Hence as temperature deviates from 37˚, R0010 changes accordingly.

4) As the R0010 transcription rate changes, the RFP concentration steady state value changes accordingly. Also, the cI steady state concentration changes.

5) To increase the visual contrast, Prm + driving GFP has been added to the system. For example, if the temperature increases from 37˚ to 42˚, the LacI dimers disassociate more readily, and [LacI dimer] decreases. R0010 transcription rate increases, resulting in higher [RFP] and [434 cI] steady state values. Since 434 cI represses Prm, the Prm transcription rate will decreases, resulting in a lower [GFP]. It is hoped that the decrease in steady state [434 cI] will indeed result in a noticeable change in [GFP], this depends on [434 cI] at 37˚ being in the linear region of a Prm + transcription rate vs. [434 cI] graph. (A way of tuning this will likely be needed – investigating currently)

Tuneable Temperature Sensor v3 (Registry 7.05 DNA-1 + Harvard Parts) [BBa_J11022]

http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6160

pBad/araC
I0500

B0034

lacI ts
J06501

B0010

B0012

lacI+pL
R0011

B0031

C0056

B0010

B0012

Prm +
I12006

B0032

E0040

B0010

B0012

LacI
R0010

B0034

mRFP1
E1010

B0010

B0012

Explanation Summary:

The same as BBa_J11022, however the parts have been moved around so that currently available parts can be used to reduce the overall number of parts (no change in functionality). Also, some minor substitutes have been made (ex. R0010->R0011) to further reduce the number of parts. For synthesis, only 8 parts have to be assembled together.

pBad/araC I0500	J06801	B0031	C0056	B0015	Prm + I12006	E0240	J04450

Bacterial Whiteboard by Hannah (w/ Minimal Available Parts) [BBa_J11031]

http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6154

tetR R0040	B0034	lacZ a E0033	B0010	B0012	lacI+pL R0011	B0034	ECFP E0022	B0010	B0012

Explanation Summary:

When lactose is added to the system, bind to lac repressor (endogenous) therefore CFP (fast degrading) produced - glow cyan. When Tet added to the system, drive transcription of lacZ beta galactosidase which hydrolyses latose and lac repressor active. Stop Glowing cyan. ***NOTE*** This system will ONLY WORK IN LAC Z- bugs!!!!!

For synthesis, only 4 parts have to be assembled together:

tetR R0040	E0433	lacI+pL R0011	E0422

Part 2: Temperature Sensor Modeling

Note: “In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription.” [http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=185]

Part 3: Assembly Schedule

Part

Day 1

Day 2

Day 3

I0500

I0500+ J06801

(I0500+ J06801)+( B0031+ C0056)

{(I0500+ J06801)+( B0031+ C0056)}+{ (B0015+ I12006)+( E0240+ J04450)}

J06801

B0031

B0031+ C0056

C0056

B0015

B0015+ I12006

(B0015+ I12006)+( E0240+ J04450)

I12006

E0240

E0240+ J04450

J04450

R0040

R0040+ E0433

(R0040+ E0433)+( R0011+ E0422)

E0433

R0011

R0011+ E0422

E0422

BBa_J11022

pBad/araC I0500	J06801	B0031	C0056	B0015	Prm + I12006	E0240	J04450

BBa_J11031

tetR R0040	E0433	lacI+pL R0011	E0422

Part 4: Part Locations

Part

Registry 7.05 DNA-1 Well

I0500

Registry 7.05	9I	Registry 7.05 DNA-2	pSB2K3	V1003
()

J06801

Registry Update	17	Harvard iGEM2005 - 1	pSB2K3	V1009
(TK Freezer)

B0031

C0056

18L

B0015

I12006

Registry Update	24	Box 5	pSB2K3	V1006
(TK -80 (1D5C))		CE100-pSB2K3-I12006
Registry 7.05	8E	Source Plate 5	pSB2K3	V1006
(TK -80)
Registry 7.05	15I	Registry 7.05 DNA-2	pSB2K3	V1006
()

E0240

16A

J04450

Registry Update	81	Box 5	pSB1A2	V1010
(TK -80 (1D5C))
Registry Update	91	Box 5	pSB1A2	V1010
(TK -80 (1D5C))

R0040

E0433

14K

R0011

E0422

11G