Team:TU Darmstadt/Achievements/Characterization
Part Characterization
Introduction
With the new bronze medal criteria Characterization being introduced for the 2019 iGEM year, teams now have the opportunity to add quantitative characterization data to already existing parts within the Registry of Standard Biological Parts. Moreover, they are able to measure parts that are relevant to their project goals. Therefore, we decided to characterize two parts that also play a significant role in our project. The first one is a Sortase A added to the registry by iGEM Stockholm in 2016 (BBa_K2144008). Since we used two mutants of the Sortase A derived from Staphylococcus aureus and focused on their in-depth characterization (Have a look at their registry pages: BBa_K3187028 and BBa_K3187016), we were excited to add another sortase variant to the collection. As a second part we characterized a tetR repressed mRFP1 (BBa_K577895). We also used the tetR repressible system to fine tune our VLP modification composition. Hence, we had the opportunity to get in contact with and experience the tetR repressible system.
All characterization data were added to the corresponding registry pages: BBa_K2144008 and BBa_K577895.
Sortase A Stockholm 2016 (BBa_K2144008)
At the start of our project, we searched for a Sortase A we could use for our project. We discovered a matching BioBrick in the registry that was submitted by the iGEM team Stockholm 2016. We added a ribosomal binding site (RBS) upstream and a T7 terminator downstream of the coding sequence. We transformed our E. coli cells and started to express this Ca2+-dependent Sortase A.
To compare the different sortases, we expressed Sortase A7M, Sortase A5M
and Sortase A from the iGEM team Stockholm 2016 (BBa_K2144008). As done with the Sortase A7M and the Sortase A5M, we purified the
Sortase A (BBa_K2144008) via fast protein liquid chromatography (FPLC)
using the ÄKTA pure (Fig. 1) by means of His-tag purification.
Figure 1: Chromatogram of the ÄKTA run. The elution of other proteins than Sortase A is shown.
Figure 2: Chromatogram of the ÄKTA run. Sortase A (BBa_K2144008) eluted as a peak at about 59 mL.
In Fig.1 it is shown that the Sortase A (BBa_K2144008) eluted at about 59 mL. Fig. 2 shows the elution of other proteins than the Sortase A. Expected size and purity of the protein were assessed by SDS-PAGE (Fig.3)
Figure 3: A SDS-PAGE with increased contrast of a triplicate of the reaction solution for the sortase-mediated reaction consisting of coat protein (CP), mCherry and Sortase A (BBa_K2144008) which were incubated for 90 minutes at 37 °C. A negative control without Sortase A (BBa_K2144008), as a triplicate, is also included. To verify the size of the Sortase A (BBa_K2144008), it was also put on the gel on its own. This functioned as a control.
The SDS-PAGE in Fig. 3 also shows the first sortase reaction performed
with the Sortase A (BBa_K2144008). On the right side a
triplicate of the purified Sortase A (BBa_K2144008) was placed to confirm it's size of approximately 15 kDa. The solvent
front appears below the Sortase A (BBa_K2144008) band. CP-LPETGG and
GGGG-mCherry were mixed and Sortase A (BBa_K2144008) was added. The
reaction was run for 90 minutes at 37 °C. As a negative
control, Sortase A (BBa_K2144008) was put on the gel by itself (triplicate on the far right). The other negative
control was of CP-LPETGG and GGGG-mCherry without the Sortase A
(BBa_K2144008) added (triplicate in the middle). As we expected to see a band at approximately 74 kDa
in the positive control, we suspected that the Sortase A (BBa_K2144008)
was not working properly. We then tried to confirm whether our
suspicion was right by performing a FRET-assay.
In order to have a definite answer, we compared our Sortase A7M to the
Sortase A (BBa_K2144008) using the FRET-pair
5-Carboxytetramethylrhodamin with a LPETG-tag (TAMRA-LPETG) and
GGGG-sfGFP (Fig. 4). We measured the reaction kinetics at
30 °C for 3 h.
Figure 4: A FRET-assay of Sortase A from Stockholm and Sortase A7M connecting TAMRA-LPETG and GGGG-mCherry with and without 10mM calcium. The ΔRFU refers to the respective negative control without each Sortase. (BBa_K2144008) and the Sortase A7M.
As visible in Fig. 4 the Sortase A (BBa_K2144008) does not show
any activity during the reaction, although the optimum of 10 mM of calcium were present in the reaction buffer. It does not seem to catalyze any
reactions over the measured time span. In comparison, Sortase A7M,
in optimal conditions without calcium, shows the expected increase in fluorescence
visible in the change of the relative fluorescence units (ΔRFU) at
514 nm over time. The ΔRFU refers to the difference between the
negative control without the respective Sortase A7M.
Had there been any activity of the Sortase A (BBa_K2144008),
it would have looked similar to the graph of the Sortase A7M, in which the
ΔRFU at 514 nm is increased due to the FRET pair being connected.
This confirms the results of the previous SDS-PAGE.
We thereby conclude that Sortase A
(BBa_K2144008) is not functional.
TetR repressed RFP (BBa_K577895)
The BioBrick BBa_K577895 is composed of a constitutively expressed tetR repressor under the control of the cat promoter (BBa_ I14033). Upstream of the tetR coding sequence, the mRFP1 gene is controlled by the p(tetR) promoter, which can be induced by addition of the antibiotic derivative anhydrotetracycline (AHT). AHT binds to the tetR repressor, which results in a decreased inhibition of the mRFP1 expression, meaning expression of mRFP1 increases when inducing with AHT.
When transforming E. coli BL21 (DE3) using BBa_K577895, the transformed culture expressed a strong red color, even without the addition of AHT. The expression of the tetR repressor seems to be too weak to properly inhibit the expression of the upstream following mRFP1 (Fig. 5).
Figure 5: BBa_K577895 transformed in E. coli BL21 (DE3) without the addition of anhydrotetracycline after cultivation overnight. The culture expresses a strong red color.
The expression of mRFP1 was measured using a SpectraMax M5E (Ex: 583 nm, Em: 607 nm) over a period of six hours with and without the addition of 200 µg/L AHT. The results are shown in Fig. 6.
Figure 6: Expression of mRFP1 (E. coli BL21 DE3) with and without the addition of anhydrotetracycline over a period of 6 hours. 0 h corresponds to the time of induction with 200 µg/L AHT at an OD600 of 0.5.
The addition of 200 µg/L AHT does not seem to have an effect on the expression of mRFP1. With and without the addition of AHT the expression of mRFP1 increases over the monitored time period.
