Difference between revisions of "Part:BBa K731700"

Revision as of 21:53, 18 September 2012

Platform for terminators analysis under the control of T7 promoter

Platform for terminators’ characterization by fluorimetric measurements and ratiometric analysis under IPTG inducible T7 promoter’s control BBa_J64997. This backbone allows a precise and consistent measure of terminator’s effect on protein expression. It contains a bicistron of fluorescent proteins: mCherry first and then A206K Venus. In between these two genes a BioBrick cloning region has been added, to insert the terminator to be tested. The comparison between the fluorescence of the two proteins with and without the terminator allows to derive some information about the terminator’s effect on protein expression.

An almost identical backbone was also submitted as BBa_K731710. To build it we used a type of insertion-deletion PCR to insert an E. coli tac transcriptional promoter and remove the T7 promoter. The combined use of BBa_K731710 and BBa_K731700 allow also to analyze any potential difference in terminators' activity due to different RNA polymerases.

Usage and Biology

Our experiments exploited an E. coli lysogen strain carrying T7 RNA polymerase and lacIq. Additionally, the cells, i.e. E. coli BL21(DE3) pLysS, also contained a plasmid encoding T7 lysozyme and chloramphenicol resistance. T7 lysozyme is a natural inhibitor of T7 RNA polymerase activity, thus reducing background expression of the target genes. The T7 RNA polymerase is behind a lacUV5 promoter.

Experimental set up We found that the emission from mVenus was much stronger than the mCherry emission, resulting in some spectral overlap. To improve the quality of the data, off-peak excitation and emission wavelengths were identified that minimized the effect. Therefore, mVenus was excitated at 485 nm (excitation peak is at 515 nm), and the emission of mVenus was collected at its maximum position, i.e. 528 nm. The maximum mCherry excitation wavelength was used (587 nm), but the emission of mCherry was read at 615 nm, as opposed to the maximum at 609 nm. The data were collected from cells that were expressed for 4 h with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). To identify optimal conditions, we screened samples that were and were not sonicated, sample dilutions to decrease the scattering of light, IPTG concentration, time of induction and time after induction. We found that the best results were obtained with induction with 0.5 mM IPTG for 3 h, followed by sonication and centrifugation. The cleared supernatant was then diluted 3-fold with phosphate buffer saline (PBS, composition) and stored overnight at 4 °C. Finally, fluorescence was measured with a Varian Cary Eclipse spectrofluorimeter as described above. The samples were left overnight at 4 °C to allow for sufficient time for the fluorescent proteins to properly mature (i.e. protein folding and chromophore formation). Each measurement was made in quadruplicate from different colonies from different transformations and from different plates.

in vitro measurements One potential reason for the increased mCherry levels could result from protection against cellular RNases. Although RNases would not explain why the effect was only observed for the T7 promoter - T7 terminator combination, we decided to test some of the constructs in a purified system that lacked the presence of nucleases. Therefore, we exploited the PURExpress, which consists of purified transcription - translation machinery including T7 RNA polymerase and E. coli ribosomes. First, the transcriptional termination efficiencies, both apparent and raw, were significantly lower in the purified, in vitro system for both T7 and E. coli terminators. This is consistent with the fact that T7 lysozyme, which was not present in our in vitro experiments, aids in transcriptional termination [*] (REF). Second, the stabilization effect was significantly diminished, suggesting that RNases or other unidentified cellular factors are involved.

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| |Excitation (nm) |Emission(nm) |- |mCherry |587 |610 |- |Venus |515 |528 |}

Sequence and Features