T1 terminator from E. coli rrnB
Since many different teams had problems using BBa_B0010, this is the same part newly submitted, confirmed by sequencing and by experience...ready to use!
The characterization of this part was done by the Trento iGEM team 2012 using the new platforms for terminator characterization that they have built and submitted to the Registry as BBa_K731700 and BBa_K731710.
Examples of Secondary Structures
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
Usage and Biology
We made some preliminary analyses to define the best procedure to characterize this terminator using BBa_K731700 and BBa_K731710. We found that the emission from A206K Venus (i.e. 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. Here is a summary of these wavelengths:
|Standard Excitation (nm)||Standard Emission (nm)||Modified Excitation (nm)||Modified Emission (nm)|
TABLE 1. Standard and modified excitation and emission wavelengths
The activity of this part was analyzed both with T7 and E. coli RNA polymerases in vivo and with T7 RNA polymerase in vitro. The activity of this terminator was measured as the ratio between the two proteins' levels using the equation found in literature for termination efficiency. Doing this measurements, however, we realized that terminators can have an effect also on mCherry expression, enhancing it. This effect was previously described Abe. As a consequence of this unexpected outcome, we defined other two parameters that are here summarized in addition to the literature definition of termination efficiency.
The parameters used to analyze the data are:
apparent termination efficiency, calculated with the equation found in literature Nojima,
raw termination efficiency, that does not consider the mCherry contribution
relative increase in the upstream gene expression,
-Vs is the A206K Venus peak’s intensity of the construct with the terminator of interest inserted in the prefix-suffix linker
-Vc is the A206K Venus peak’s intensity of the control construct without intervening terminator
-Cs is the mCherry peak’s intensity of the construct with the terminator inserted
-Cc is the mCherry peak’s intensity of the control construct
In vivo measurements:
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.
FIGURE 1. E.coli terminator's effect on protein expression with two different RNA polymerases
The data shown in figure 1 were acquired in two different days. For each day 4 different replicates were measured at different times. Briefly, BL21(DE3)pLysS were grown in 10 mL of LB until an OD of 0.6 was reached and induced with 0.5 mM IPTG. After 3 hours of induction, 4 separate aliquots of 1 mL were taken and sonicated 3 times for 10 seconds at intervals of 30 seconds. After sonication the samples were diluted 1:2 with PBS 1X directly into a cuvette and incubated overnight at 4°C. Fluorescence measurements were taken with a Cary Eclipse Varian fluorimeter with a window ranging from 450 nm to 700 nm, a slit of 5nm, a voltage of 570V for the characterization with T7 polymerase and of 520V for the measurements with E. coli RNA polymerase.
In vitro measurements:
We performed the in vitro analysis only with the T7 RNA polymerase.
FIGURE 2. E.coli terminator's effect on in vitro protein synthesis with the T7 RNA polymerases
Cell free measurements were done with the PurExpress kit from New England Biolabs, using 250 ng of DNA previously purified by ethanol precipitation, following the protocol suggested by the manufacturer. Measurements were done with a PTI fluorimeter.
We submitted also this Part inside both BBa_K731700 (T7 promoter backbone) and BBa_K731710 (tac promoter backbone) as, respecitively, BBa_K731701 and BBa_K731702. We hope that this could be helpful for anyone who want to verify and further delve into our results.
More information about the procedure used, the results obtained and our considerations can be found in the Trento iGEM 2012 wiki page.
- Nojima pmid=16379390
- Abe pmid=9150882
Functional Parameters: Austin_UTexas
Burden Imposed by this Part:
Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.
This functional parameter was added by the 2020 Austin_UTexas team.
|forward_efficiency||0.976 +/- 0.005 with T7 RNAP (T7 promoter) 0.985 +/- 0.001 with E. coli RNAP (Ptac)|