IGEM:IMPERIAL/2007/Resources/Presentations/120907

CBD Re-focus (12th Sep 2007)

Introduction

This report aims to elucidate the limitations of our design and fabrication with relation to our original specifications. Essentially, the final objective is to be able to re-focus the project in a way that is feasible.

Original Specs

The original specifications of our project was defined as such:

• Report cumulative temperature exposure of food
• Easy to use with visual signals, + time response

• Improve accuracy of other attempts by meat industry
• System must be stable for length of shelf-life, how long is that ?
• No contact with food
• + range of operating temperatures

Design

The design of the system consists of two components -

• Reporter system - pTet constitutive promoter + Fluorescent reporter
• Chassis - Cell Free systems

The design of the system consists of three components -

• Tempreature Exposure Reporter - pTet constitutive promoter
• Visual reporter - Fluorescent reporter
• Chassis - Cell Free systems (+ packaging ?)

Limitations of the Design

Reporter system

• GFP is not a visual signal; unless DsRed-Express delivers and functions as intended.
  • When DsRed-Express is supposed to be delivered ? When the construct is supposed to be built ?
• Reporter system stops working at 10oC.
  • Do you have any data showing that the system works above 10oC, and below room temperature ?
• Non-linear kinetics in relation to temperature.
  • Non-linear is ok, non-monotonic is a problem.

The last two points may be more or less due to the limitations of our chassis. Explain, it is not obvious to me

Chassis

• Papers suggest that E. coli halts protein synthesis at 7.8°C. In vitro ? In Vivo? which papers ???)
• Coupled transcription-translation machinery is halted at the initiation of translation both in vivo and in vitro. This is due to a few factors:
  • Increased/induced stability of secondary RNA structure. Have we checked the possible secondary structures of our specific construct ?
  • Reduced coupling of 70S ribosomal subunits.
  • No induced response of cold-shock genes (due to removal of E. coli DNA). These include CspA, IF2 and other protein chaperones. You mean that the folding of the protein produced could be disrupted ? Because the rest of the gene expression machinery is already folded.
• In addition, the optimal synthesis of proteins in vitro is 25°C. Further increase in temperature leads to counter-production of proteins as RNase begins to break down mRNA at a faster rate than transcription rates.

Papers (which paper ?)however describe the build-up of nontranslating ribosomes and mRNA within in vivo and in vitro systems, although ribosomal proteins levels appear normal.

Indeed there is a 'cold-shock' adaptation model in which protein synthesis is halted at initiation of ribosomal translation near 15°C, resulting in the induction of proteins that aid in resuming translation before re-commencement of protein synthesis when the ribosome and other factors are more 'adapted' to cold-shock translation.

A good point to note is also that the elongation process for both transcription and translation rates are proportional to temperatures up to 44°C.

What does this mean

From the perspective of our specs, we have accomplished the 2 specs, but falter in the first three. While DsRed-Express should give us the visual signal required, it is unlikely the design, without subjecting to changes, would meet the first requirement of reporting the cumulative thermal exposure of food. This is because the minimal operating range for our system should be 4oC, not 7.8oC, or whatever the operating range our experiments provide. This also suggests the likelihood that if our device were to proceed as such, it would be more of a threshold device.

• A threshold device would mean that as soon as you reach a given temperature, you are reporting the event. However, with the construct presented here, the temperature can have a spike at 30oC for 5 seconds, and the device will not record, or report the event. You would need to have a very sensitive toogle-switch, for example, to claim that you have a threshold detector

Notes on Re-focus attempt

• Market as a threshold device as opposed to a cumulative one?
• Change specifications such that it only reports temperatures from minimal temperature range onwards
• RNA as a quantifying product? (RNA synthesis is reduced to 20% of the transcription rate of. 25oC at 5oC)
• Others.

References

1. Broeze RJ, Solomon CJ, and Pope DH. Effects of low temperature on in vivo and in vitro protein synthesis in Escherichia coli and Pseudomonas fluorescens. J Bacteriol. 1978 Jun;134(3):861-74. DOI:10.1128/jb.134.3.861-874.1978 |
2. Anderson WA. Kinectics of beta-galactosidase synthesis in Escherichia coli at 5 C. J Bacteriol. 1975 Mar;121(3):907-16. DOI:10.1128/jb.121.3.907-916.1975 |
3. Jones PG, VanBogelen RA, and Neidhardt FC. Induction of proteins in response to low temperature in Escherichia coli. J Bacteriol. 1987 May;169(5):2092-5. DOI:10.1128/jb.169.5.2092-2095.1987 |
4. Herendeen SL, VanBogelen RA, and Neidhardt FC. Levels of major proteins of Escherichia coli during growth at different temperatures. J Bacteriol. 1979 Jul;139(1):185-94. DOI:10.1128/jb.139.1.185-194.1979 |
5. Thieringer HA, Jones PG, and Inouye M. Cold shock and adaptation. Bioessays. 1998 Jan;20(1):49-57. DOI:10.1002/(SICI)1521-1878(199801)20:1<49::AID-BIES8>3.0.CO;2-N |
6. Gualerzi CO, Giuliodori AM, and Pon CL. Transcriptional and post-transcriptional control of cold-shock genes. J Mol Biol. 2003 Aug 15;331(3):527-39. DOI:10.1016/s0022-2836(03)00732-0 |
7. Narberhaus F, Waldminghaus T, and Chowdhury S. RNA thermometers. FEMS Microbiol Rev. 2006 Jan;30(1):3-16. DOI:10.1111/j.1574-6976.2005.004.x |
8. Storch D and Pörtner HO. The protein synthesis machinery operates at the same expense in eurythermal and cold stenothermal pectinids. Physiol Biochem Zool. 2003 Jan-Feb;76(1):28-40. DOI:10.1086/367945 |