Natasha Tuskovich

Friday, June 12, 2009

E. coli Bio-Thermometer

Introduction

A biological thermometer would ideally be a simple, accurate and easily observable register of the organism’s environment. A basic version is comprised of E. coli cells that respond to differences in temperature by producing different fluorescent proteins. It is essentially cells that can appear blue or green or red depending on the temperature they are exposed to.

The TU Delft 2008 team has produced a number of RNA thermometers, with varying levels of effectiveness. They retrieved natural RNA thermometers from three species, then sequenced and redesigned them to test for temperature variability range. They have produced temperature-sensitive RNA biobricks that include the ribosome binding site and can prevent the SD sequence for the next part from being read. This is useful because it provides temperature control for any output.

Figure 1. General Temperature-Sensitive RNA Thermometer

Combining this with activators and repressors produces temperature-dependent responses. For the system to turn reporters on and off, multiple activators and repressors are used to provide the different states. For instance, going from a low to a high temperature, the lowest state (cyan), must be turned off and stay off; the middle state (green) must be both turned on and then off, and the highest state (red), must be turned on only during high temperatures. Ideally only one colour would be present at any stable state.

Using two of the RNA thermometers developed by TU Delft 2008, we can produce a system with three different states spaced along a temperature gradient. Below 32 oC, cyan fluorescence protein (CFP) is produced under the control of a LacI promoter. Both the LuxR activator for green fluorescence protein (GFP) and the LasR activator for red fluorescence protein (RFP), are unable to be translated at this low temperature, and thus the system glows blue. Above 32 oC but before reaching 37 oC, two things change. The LacI repressorcan now be translated thereby shutting CFP off. As well, the activator for GFPis produced changing the system to green. After 37 oC, the cI repressor controlling GFP, is translated stopping production of GFP, and the LasR activator is also produced and turns on the RFP genes for the final red state.

Figure 2. Below 32 oC – Cyan Active

CFP is produced with the LacI promoter, but neither GFP nor RFP are activated yet.

Figure 3. Between 32 oC and 37 oC– Green Active

The LacI promoter for CFP is blocked and GFP is activated by the LuxR activator, but RFP is still not activated.

Figure 4. Above 37 oC– Red Active

Both CFP and GFP are blocked by repressors and the LasR activator has started RFP production.

Parts

Figure 5. CFP Plasmid

The CFP genes on this plasmid are under control of LacI, while the LacI repressor is under control of a constitutive promoter and a hairpin loop that opens at 32oC. CFP is produced until the temperature reaches 32 oC, whereupon the LacI repressor is now produced and stops production of CFP. This plasmid is the most likely to work.

Figure 6. GFP Plasmid

A LuxR/lambda cI promoter regulates GFP production, while a constitutive promoter controls both the LuxR activator and the cI repressor, also regulated by a 32oC hairpin loop and a 37 oC hairpin loop respectively. Before reaching 32 oC, the LuxR activator is not produced and thus GFP is not produced until that temperature. When the temperature reaches 37 oC, the cI repressor is now present and stops GFP production.

Figure 7. RFP Plasmid

RFP production is controlled by a LasR promoter, and the LasR and LasI activators are controlled by a constitutive promoter and a 37 oC hairpin loop. This means that RFP is only turned on after reaching 37 oC, when the activators are able to be produced.

Table 1. Temperature Responsive Regulators

BBa_K115017 / RNA thermometer / Becomes active at 32 oC
BBa_K115020 / RNA thermometer / Becomes active at 37 oC

Figure 8.Temperature-Sensitive Hairpin Loops

(BBa_K115020 is the hairpin loop of BBa_K115001.)

BBa_K115017, and BBa_K115020 were chosen from TU Delft 2008’s different thermometers because the first was shown to be the most successful with a clear response when heated above 32 oC; and the second is intended to be indicative over 37 oC, which holds interest as the human body temperature.

Table 2. Flourescent Protein Reporters

BBa_E0020 / Engineered cyan fluorescent protein / Used to indicate below 32 oC
BBa_E0040 / Green fluorescent protein / Used to indicate between 32 and 37 oC
BBa_E1010 / Red fluorescent protein / Used to indicate above 37 oC

Fluorescence proteins were chosen because the intention of the project is to have a visible display of the temperature felt by the cells. In addition, this type of response is assayable and simple.

Table 3. Promoters and Regulators

BBa_R0011 / LacI regulated / (Induced by IPTG) / Repressed by LacI: BBa_C0012
BBa_R0065 / LuxR/lambda cI regulated / Activated by LuxR: BBa_C0062 / Repressed by cI: BBa_C0051
BBa_R0079 / LasR/ LasI regulated / Activated by LasR: BBa_C0079, and LasI: BBa_C0078 / (Not repressed)
BBa_K137029 / Constitutive / (Not activated) / (Not repressed)

Table 4. General Parts

BBa_B0034 / Ribosome binding site
BBa_B0030 / Ribosome binding site
BBa_B0015 / Double terminator consisting of BBa_B0010 and BBa_B0012
BBa_P1010 / ccdB death gene
pSB1C3 / Construction vector with chloramphenicol resistance
pSB1A2 / Plasmid with ampicillin resistance
pSB1AT3 / Plasmid with ampicillin and tetracycline resistance

Table 5. Pre-combined

BBa_I13601 / CFP reporter repressed by LacI / BBa_R0011-BBa_B0034-BBa_E0020-BBa_B0015
BBa_E0840 / GFP reporter / BBa_B0030-BBa_E0040-BBa_B0015
BBa_S03885 / RFP reporter activated by LasR / BBa_R0079-BBa_B0034-BBa_E1010

Assembly

3A assembly is designed to use three-way ligation and both positive and negative selection to minimise incorrect assemblies. 3A assembly stands for three antibiotic assembly, as the three starting plasmids all have different antibiotic resistance markers. The construction vector (red) contains ccdB (black block arrow) and the EcoRI and PstI restriction sites. The blue plasmid contains the prefix part (pink), and the green plasmid contains the suffix part (yellow). These two plasmids can have the same or different resistance markers, as long as they are distinct from the construction vector. All these BioBrick parts are standardised to contain these restriction sites and an antibiotic resistance marker making this an appropriate method for the whole project. There are also an assortment of construction vectors with different antibiotic resistance markers enabling simple changes from part to part as only up to three antibiotics will be necessary at one time.

EcoRI (E) and PstI (P) are used to cut the construction vector, removing the ccdB gene and leaving E and P open ends. EcoRI (E) and SpeI (S) are used to cut the prefix plasmid, and XbaI (X) and PstI (P) are used to cut the suffix plasmid. These pieces of DNA can then be ligated together and transformed. A mixed site (M) is left between the combined parts. A plate supplemented with the red antibiotic is used to grow the transformed cells. For the red vector, a ccdB resistant strain such as DB3.1 must be used.

Figure 9. 3A Assembly

Digested parts before ligation.

Figure 10. 3A Assembly

Purified plasmid fragments ligated into the construction vector.

Methods

This procedure is taken from the open wetware page:

Construction Procedure

  1. Miniprep the two parts and construction plasmid. e.g.

Prefix part BBa_K137029 comes on a pSB1A2 plasmid with ampicillin resistance

Suffix part BBa_K115017 comes on a pSB1AT3 plasmid with ampicillin and tetracycline resistance

Construction vector pSB1C3 is a plasmid backbone with chloramphenicol resistance

  1. Digest the two parts and destination vector with the following enzymes
  2. Prefix part (BBa_K137029) with EcoRI and SpeI
  3. Suffix part (BBa_K115017) with XbaI and PstI
  4. Construction vector (pSB1C3) with EcoRI and PstI
  5. Purify the restriction digest.
  6. For parts < 200 bp in length, do a Knight:Micropure EZ and Microcon purification (or alternatively an Ethanol precipitation of small DNA fragments). All other parts and the construction vector can be purified using a QIAquick PCR purification kit or Qiagen Minelute PCR Purification Kit (elutes in a smaller volume giving greater concentration of the DNA).
  7. Ligate the two parts and construction vector together. (Endy lab protocol)
  8. Transform the ligation product. (See also Electroporation or another chemical transformation.)
  9. Analyze the transformation with single colony PCR followed by agarose gel electrophoresis.
  10. In rolling, large scale assembly, this step is omitted.
  11. Miniprep clones that generated a band of the appropriate size.
  12. Sequence the clone.
  13. Repeat for all of the parts to form three complete plasmids.
  14. Assemble all three plasmids together to produce cells with all three responses.

The antibiotic resistance of the different parts helps to ensure that only the construction vector backbone plasmid is selected for, while the ccdB gene prevents unaltered construction vectors from continuing. These plasmids would be finally assembled together in a single cell. It may be advantageous to switch from high copy number assembly plasmid backbones, used while combining the parts, to low or medium copy backbones for the final product.

The individual plasmids should be tested first on their own to determine their individual effectiveness. This would also allow us to more easily form BioBrick parts for these systems. The working parts would then be combined to produce cells with multiple temperature reactions.

Testing Procedure

  1. Grow each plasmid individually in cultures at 30 oC, 35 oC, and 38 oC (nine samples).
  2. Grow the combined cells in cultures at 30 oC, 35 oC, and 38 oC (three samples).
  3. Grow control cultures to account for system biases.
  4. Assay for fluorescence proteins.

Discussion

The largest issue that might be a problem is that the 37oC RNA thermometer part (BBa_K115020) has not been shown to be effective. If it is in fact ineffective this would mean that only the CFP plasmid would work entirely correctly. The GFP plasmid would either never be repressed (still useful by producing a working change in temperature at 32oC), or would be always repressed (no GFP ever produced). The RFP plasmid would also suffer similarly, either always on or always off, serving no purpose. In this situation, we would want to assemble a system involving only the 32 oC dependent aspects; this system would only have one change, from blue to green at 32 oC.

Other factors that might hinder the overall visible result include, if the fluorescence proteins take too long to show visible results; if the old fluorescence proteins take too long to disappear after a change in temperature; if leakiness in the promoters leaves too much background colour, muddying the indicator; and if the repressors and activators do not produce a great enough change in fluorescence to be observable. Depending on these factors the system may be most accurate when intended for use at only one temperature – a situation that would then have proof of being at a constant temperature within a certain range as long as the expressed colour was relatively pure. Mixed or muddy colouring would indicate inconstant temperature.

Conclusion

This presents a procedure for developing a bacterial thermometer with three distinct states using two RNA temperature switches. All parts are from the iGem Parts Registry and are suitable to assemble and use together. The 3A assembly method is appropriate for these parts and can be implemented with a combination of relatively standard processes and techniques.

References

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