IGEM:IMPERIAL/2007/Projects/Cell by date/Design

Cell by Date: Design

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Summary

Cell by Date has three levels of complexity and we hope to implement these levels in separate chassis: E. coli, in vitro and in veso. These chassis are being characterized as one of ICGEMs sub-projects. The table below outlines which levels which hope to implement Cell by Date in.

Chassis	Phase 1	Phase 2	Phase 3
E.coli	X
In-Vitro	X	X	X
In-Veso	X	X	X

Phase 1: Initial Testing

Phase 1 involves the testing of various simple constructs to confirm that there is gene expression in vivo and in vitro. DNA constructs are as summarized below:

Type of Promoter	Test Constructs	Notes
Constitutive T7 Promoter	PT7 promoting GFP	Entire construct is currently available in Biobricks Registry: BBa_E7104 (available), in pSB1A2 (AmpR) pT7 can be used with T7 RNAP in vitro and in veso, and it gives constitutive expression.
Constitutive E.coli Promoter	Ptet</sub promoting GFP	Entire construct is currently in Biobricks Registry: BBa_I13522 (available), in pSB1A2 (AmpR) pTet can be used with E.coli RNAP in vitro and maybe in veso, and it gives constitutive expression.
Inducible E.coli Promoter	PBad promoting GFP	pBad promoter and GFP available: BBa_J5528 (available), in pSB2K3 (KanR) Need Ara C regulator protein in vitro and in veso. Ara C available with the RBS:BBa_S03550 pBad can be used with E.coli RNAP in vitro, and it is inducible by arabinose. To be used in veso, arabinose transport proteins have to be inserted in the phospholipid bilayer of the vesicles.
Constitutive E.coli Promoter	PcI promoting GFP	Entire construct is currently available in Biobricks Registry: BBa_J07037 (avaiable), in pSB2K3 (KanR) pcI gives constitutive expression.
Constitutive E.coli Promoter	pBad promoting GFP and pcI promoting araC	All parts are currently available in the Biobricks Registry. pcI promoter: BBa_R0051 (available), in pSB1A2 (AmpR) Arabinose C regulator protein with RBS: BBa_S03550 (available), in pSB1AC3 (A+CR) Stop codon: BBa_B0015, in pSB1AK3 (A+KR) pBAD with GFP: BBa_J5528, in pSB2K3 (KanR)

We are not interested in a lot of details, but rather just trying to see the most suitable promoter. The promoter construct, together with the inducer required (if the promoter is inducable) will be mixed together and the levels of fluorescence will be monitored over time in different chassis.

• 3 repeats to be done for each test
• Positive control: Purified GFP in E.coli, and then in the cell extract
• Negative control: Cell extract without any DNA insert or GFP

The best promoter sequence is defined as such:

• Fastest expression of GFP
• Highest detection of fluorescence

We preferably want to use a constitutive promoter for Cell by Date. If all constitutive promoters are found working then we will need to chose one of them. This decision can be made based on their levels of activity (the higher, the better) and their operating temperature range. This needs to be as close as possible to the CBD operating range of 4°C - 37°C

Phase 2: Characterizing specific Construct

Once the best promoter has been chosen, we would then proceed to characterize it in the in vitro systems. Several variables will be tested for, including:

• Test for operating temperature range: 4°C, 15°C, 25°C, 37°C, 45°C, 60°C.
• Life span of the system

Fluorescence level readings will be taken every 15 min intervals, with triplicates for each sample at a particular temperature. Determining the rate of fluorescence at each temperature would also provide preliminary data for modelling analysis.

The system is then subjected to temperature changes and the effect on fluorescence levels measured:

• Subject system to temperature changes and measure effect on fluorescence levels
• Type of gradients: Steep/ Gentle/ Pulse
• Temperature increment: from 4°C to 25°C
• Apply the temperature increment only after the GFP levels have reached steady state

Problems

• The commercial cell extracts come in 50µl, while the wells in the fluorometer plates can hold up to 200µl.
• Water evaporates when fluorescent readings are taken.

Phase 3: Testing/Validation of Modelling Analysis

Ideally, phase 2 would provide the results necessary for constructing our model for the system. The model should also be able to accurately predict the outcome of our temperature effects and verified through experimental testing. We would then obtain the thermal exposure device that achieves the specifications that we have defined for the project.

Phase 2 and 3 is to be repeated using DsRed-Express as the reporter. This is subjected to the arrival of its gene sequence, and its preparation and performance overall as a better reporter (due to its stability and quicker detection time).

Considerations for DsRed-Express

• When Dsred Express gene sequence arrives, clone and express it.
• Draw up a calibration curve of DsRed-Express.
• Positive control: DsRed-Express in cell extract
• Negative control: Cell extract only