Difference between revisions of "Part:BBa B0010:Experience"

iGEM Athens 2019

We attempted to measure the efficiency of the terminator by cloning it into the pSB1A10 plasmid and culturing the bacteria for 24 hours (37 degrees Celsius, 200RPM, 0.1% arabinose, 100 µg/mL ampicillin). The culture was pelleted and the pellet was resuspended in 1mL of 1X PBS. From that, 10 μL were diluted in 990 mL of 1X PBS to yield approximately 5 million cells. For FACS, we used BD FACSCanto™ II. During FACS, a low flow rate was used. We used the following excitation and emission wavelengths: GFP: excitation = 488 nm, emission = 510 nm ; RFP: excitation = 488 nm, emission = 584 nm.

FACS measurement. The terminator was named ABC terminator for our project.

The efficiency is measured through the following formula: Termination efficiency = 1 -{[RFPterm/GFPterm]/[(RFPcontrol/GFPcontrol)mean]}. This measurement showed that the efficiency of the terminator is 0%. We also measured the terminator efficiency using the GFP+RFP+ population and the formula: Terminator efficiency = 1 - (Intensity of GFP+RFP+term/Intensity of GFP+RFP+control). This measurement showed that the terminator efficiency of the terminator is 7%.

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

Trento iGEM team 2012

This part was successfully extracted from the distribution kits 2012 and 2011. However, it was not confirmed by restriction digestion nor PCR. We were able to amplify it by PCR from BBa_E0840, that contains the double terminator BBa_B0015, subcloned it into pSB1C3 and confirmed by sequencing. We re-deposited rrnBT1 terminator well-characterized and well-functioning as BBa_K731722.

The characterization of this part was done using the platforms for terminators' characterization BBa_K731700, BBa_K731710 with both T7 and E. coli RNA polymerases in vivo and with T7 RNA polymerase in vitro.

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 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:

TABLE 1. Standard and modified excitation and emission wavelengths. We adopted these modified excitation and emission wavelengths for both in vivo and iv vitro measurements.

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,

where

-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 can be found in the iGEM Trento 2012 wiki page.

References

<biblio>

1. Nojima pmid=16379390
2. Abe pmid=9150882

</biblio>