Team:TU Dresden/Measurement

Short Description

In bacteria, protein secretion is mainly orchestrated by the Sec Pathway via Signal Peptides (SP), which are located at the N-terminus of secreted proteins. The secretion efficiency is not determined by the sequence of the SP alone, but instead is the combined result of an SP with its specific target protein. This necessitates establishing efficient screening procedures to evaluate all possible SP/target protein combinations. We developed such an approach for our Signal Peptide Toolbox, which contains 74 Sec-dependent SPs. It combines combinatorial construction with highly reproducible, quantitative measurements. By applying this procedure, we demonstrate the secretion of three different proteins and succeeded in identifying the most potent SP-protein combination for each of them. This thoroughly evaluated measurement tool, in combination with our SP toolbox (fully available via the partsregistry) enables an organism-independent, straightforward approach to identifying the best combination of SP with any protein of interest.

Background

Over the course of the last decades the quality, amount and spectrum of heterologous (and recombinant) proteins has drastically increased and therefore the need for techniques to easily express and purify these proteins has emerged. We find such proteins as ingredients of detergents (proteases), medical treatments (insulin) or food and beverage products (amylases). Simply put, heterologous proteins are ubiquitously present. [1]

In order to tackle this demand we chose to apply the genetic tools of the model organism Bacillus subtilis. It is already one of the most frequently used hosts for overproduction of proteins throughout academia and industry because of its tremendous capacity to secret proteins, which can be exploited to increase the overall yields.

B. subtilis has four different secretion pathways, however the majority of proteins are being secreted via the general Sec pathway (Figure 1). This pathway has been identified playing a crucial role in protein secretion as common element among all domains of life [2]. In the Sec pathway, the secretion of proteins into the surrounding supernatant is orchestrated by signal peptides (SP). These SPs are composed of approximately 60 to 180 nucleotides and they are located upstream of the protein to be secreted. Intracellularly, the SP is translationally fused to the specific protein but cut off during the membrane translocation process releasing the protein into the supernatant without the signal peptide attached to it. [3]

A scheme explaining the Sec pathway of Bacillus subtilis.
Figure 1: The Sec pathway of B. subtilis. 1 The protein (blue) and its N-terminally fused SP (red) are ribosomally synthesized (purple). 2 A Signal Recognition Particle (orange) transports the protein to the membrane. 3 At the membrane, the translocation complex (light and dark green) takes over the protein. 4 During the translocation process, the protein is hold in the translocation complex but the SP is cut off by a peptidase (grey). 5 Afterwards, the protein is released into the supernatant where it reaches its native fold and the SP is degraded.

Up to date, approximately 170 SPs belonging to the Sec pathway of B. subtilis have been annotated but unfortunately, no correlations on sequence levels have been identified that link efficient protein secretion with a distinct SP. [4]

As part of our project EncaBcillus, we aimed at creating a platform for heterologous protein overexpression combined with their efficient secretion without releasing any cells into the environment. Hereby, we encapsulated our model organism B. subtilis in the Peptidosomes which serve as a new innovative method to keep the producing bacteria separated from the desired compounds. (For more details see Peptidosomes.)

To address the vision of creating this new platform, we first wanted to establish a fast and easy screening procedure to evaluate all combinational possibilities of each SP with a protein of interest (POI) – The Signal Peptide Toolbox.

As SPs of B. subtilis have been successfully adapted to GRAM-positive [5] and GRAM-negative bacteria [6], the Signal Peptide Toolbox, as an organism-independent genetic measurement tool, can be applied to any bacterial host. Every future iGEM team will be able to use this fully partsregistry availabe tool to increase their protein secretion.

Design

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Table 1: Submitted Sec SPs of B. subtilis.
 AmyE   AspB   BglS   Bpr   CccA   CitH   Csn   DacB   DacF   DltD 
Epr FliL FliZ GlpQ LipA LytB LytC LytD LytR Mdr
Mpr NprE PbpX Pel PelB PenP PhoA PhoB PhrA PhrC
PhrF PhrG PhrK RpmG SacB SacC SleB SpoIID SpoIIP SpoIIQ
SpoIIR TyrA Vpr WapA YbbC YbbE YbbR YbdG YbdN YbfO
YbxI YdbK YdhT YdjM YdjN YfhK YfjS YfkD YfkN YhcR
YhdC YhfM YhjA YjcN YjdB YjfA YjiA YkoJ YkvV YkwD
YlaE YlbL YlqB YlxF

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Signal peptide primer collection table

Results

So far, no direct correlation between the perfect combination of signal peptide and downstream sequence to gain optimal secretion levels is known. Thus, the problem of having to create one clone per combination of SP and protein of interest remains. Therefore, we created the so-called Signal Peptide Mixes (SPMs), a set of libraries with each containing equal concentrations of up to twenty distinct SPs which can be easily enriched via multi-template PCR. The amplified SPs can then be combined with our Signal Peptide Evaluation Vector (SP-EV) and the gene of interest (For more details see our protocol: “Cloning with the Signal Peptide Evaluation Vector”).

Figure 3: SPM with twenty SPs. All twenty distinct SPs could be amplified using individual primers for each SP.
Figure 2: SPMs with different amounts of SPs. A SPMs with 53 distinct SPs. B SPMs with 20 distinct SPs.

In a first approach, we evaluated the multi-template PCR by varying the number of different SPs in one mix. Our aim was to amplify all SPs equally for the downstream cloning procedures. To test this, a SPM subset containing 53 SPs, was amplified using the RFC10 prefix and suffix as primers, we expected a band at around 100-200 Bp (size range of the SPs). Unfortunately, we also observed a second dominant band (at about 250 Bp) (Figure 2, A), leading to the conclusion that a subset of 53 SPs was not suitable for our purposes.

Form this we decided to reduce the number of different SPs to 20 and also to increase the primer concentrations. These improvements lead to a specific amplification of our SPs (Figure 2, B). To evaluate, if all 20 SPs were indeed amplified, we conducted a second PCR using the first PCR as template with specific primers for each SP of the original SPM. We could show that all 20 distinct SPs of the SPM subset were amplified during the first PCR (Figure 3). Therefore, we decided to split up all provided SPs into subset-mixes of each containing up to 20 SPs max.

All 74 SPs which we do provide were therefore assigned to four distinct SPM subsets. The table below gives an overview about the assignment of the SPs.

Table 2: SPM subsets a-d of all submitted Sec SPs.

SPM subset a

SPM subset b
AmyE AspB BglS Bpr CccA CitH Csn DacB DacF DltD LytR Mdr Mpr NprE PbpX Pel PelB PenP PhoA PhoB
Epr FliL FliZ GlpQ LipA LytB LytC LytD PhrA PhrC PhrF PhrG PhrK RpmG

 

SPM subset c

SPM subset d
SacB SacC SleB SpoIID SpoIIP SpoIIQ SpoIIR TyrA Vpr WapA YdjN YfhK YfjS YfkD YfkN YhcR YhdC YhfM YhjA YjcN
YbbC YbbE YbbR YbdG YbdN YbfO YbxI YdbK YdhT YdjM YjdB YjfA YjiA YkoJ YkvV YkwD YlaE YlbL YlqB YlxF

After various adjustments to enhance the Signal Peptide Toolbox’ applicability, we established the following two protocols:

  1. Cloning with the Signal Peptide Evaluation Vector (SP-EV)
  2. High through-put screening procedure

The provided Standard Operating Procedure (SOP) for cloning with the EV-SP is a modified version of the SOP for cloning with the EV. This SOP was tailored to explain the random integration of the SPs using the cloning host E. coli as the EV was evaluated in the course of our Signal Peptide Toolbox.

SOP

Example

1

Digest both, the provided EV and the new promoter using the restriction enzymes EcoRI and BsaI.

Successfully transformed E. coli colonies on X-Gal containing agar plates stay blue. But on not X-Gal containing agar plates, successfully transformed E. coli colonies stay red.

For our EV, we did not replace the xylose inducible promoter PxylA.

2

Digest both, the EV from step 1 and the new gene of interest using the restriction enzymes NgoMIV and PstI.

Successfully transformed E. coli colonies on X-Gal containing agar plates become red. But on not X-Gal containing agar plates, successfully transformed E. coli colonies stay red.

We inserted the gene amyE which encodes for the alpha-Amylase of B. subtilis.

3

Digest the EV from step 2 using the restriction enzymes BsaI and NgoMIV.

4

Aliquot all SPs to 0.5ng/µL and distribute up to 20 SPs to each SPM subset.

The SPs were distributed as shown in Table 2.

5

Amplify all SPM subsets via standard PCR using the RFC10 prefix as forward primer and suffix as reverse primer.

We recommend the following adjustments:
- 1.5 μM end concentration for each primer
- 1.5 ng SPM subset per 100 µL reaction
- 30 amplification cycles to avoid artifacts

We used the following PCR setting:

Mix

Programme*

66 μL MiliQ
20 μL 5x Q5 Buffer
15 μL forward primer
15 μL reverse primer
3 μL SPM subset
2 μL dNTPs
1 μL Q5 Polymerase**

98°C
98°C
63°C
72°C
72°C
15°C

30 s
10 s
40 s
20 s
2 min
pause

*repeat cycle steps two to four for thirty times
**purchased from NEB

6

Digest all SPM subsets from step 5 using the restriction enzymes XbaI and AgeI.

7

Distribute equal amounts of digested EV from step 3 to each digested SPM subset and fuse the SPs into the EV.

For our ligation, we chose to fuse 12.5 μL of EV with 7.5 μL of SPM subset.

8

Use the ligation mix from step 7 and directly transform your bacteria.

We performed the ligation reaction over two days at room temperature prior to our B. subtilis transformation.

We recommend to purify all digested products except the digested SPM subsets via gel extraction. Regarding the SPM subsets, we recommend to purify them via standard DNA cleanup procedure.

References

[1] van Dijl J. M. and Hecker M. (2013) Bacillus subtilis: from soil bacterium to super-secreting cell factory. Mircobial Cell Factories 12, 3 (1-6).
[2] Papanikou E., Karamanou S. and Economou A. (2007) Bacterial protein secretion through the translocase nanomachine.. Nature Reviews Microbiology 5, 11 (839-851).
[3] Fu L. L., Xu Z. R., Li W. F., Shuai J. B., Lu P. and Hu C. X. (2006) Protein secretion pathways in Bacillus subtilis: implication for optimization of heterologous protein secretion. Biotechnology advances 25, 1 (1-12).
[4] Brockmeier U., Caspers M., Freudl R., Jockwer A., Noll T. and Eggert T. (2013) Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. Journal of molecular biology 362, 3 (393-402).
[5] Hemmerich J., Rohe P., Kleine B., Jurischka S., Wiechert W., Freudl R. and Oldiges M. (2016) Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum. Mircobial Cell Factories 15, 208.
[6] Pechsrichuang P., Songsiriritthigul C., Haltrich D., Roytrakul S., Namvijtr P., Bonaparte N., Yamabhai M. (2016) OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. Springerplus 5, 1200.