Part:BBa_K3487013

Tachyplesin I multimers with GST-tag

The GST-tag has the size of 220 amino acids (roughly 26 KDa). It is fused to the N-terminus of the Tachyplesin I multimers BBa_K3487001and includes a PreScission Protease domain for cleavage of the GST tag during protein purification.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 673
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 85
  

2020 SZPT-CHINA

Description

Tachyplesin I（TP-1）is a cysteine-rich antimicrobial peptide derived from the hemocytes of horseshoe crabs. It is a broad-spectrum antibacterial peptide with good antibacterial function. TP-1 is composed of only 17 amino acids. Considering the difficulty of expressing short peptides, we use endoglutamate as the restriction site to repeat the TP-1 four times in series to form a TP-1 multimers（TP-1M）. In addition, in order to facilitate subsequent protein purification, we fused TP-1M with a GST tag. Through this strategy, we successfully expressed and purified GST-TP1M.

Result

Expression of GST-TP-1M in E. coli BL21

The recombinant strain E. coli BL21 (DE3) with pGEX-4T-2-TP-1M vector was induced by IPTG for 6 hours and then collected for SDS-PAGE and Western blot analysis. The results of SDS-PAGE were shown in Fig.3.

After IPTG induction, the E. coli BL21 transferred with pGEX-4T-2 vector produced 26kDa GST protein and E. coli BL21 transferred with pGEX-4T-2-TP-1M produced 36kDa protein matched well with our GST-TP-1M fusion protein.In contrast, Non-induced E. coli BL21 cells did not express GST or GST-TP-1M.Western blot results showed 36kDa fusion protein carry GST tag. The results showed that the constructed tandem TP-1M could be expressed in the fusion expression system.

Fusion protein purification results

Recombinant bacteria were induced to express, and the cells were collected, ultrasonically broken, and the inclusion bodies were dissolved. After affinity chromatography, the fusion proteins GST-TP1M was obtained. The SDS-PAGE electrophoresis detection result is shown in the Fig.4.

Characterization by 2021iGEM_Shanghai_United_HS

Improvement of an existing part

Compared to the old part BBa_K3487013, we design a new part BBa_K4030007 which contains the araBAD promoter of the L-Arabinose operon of Escherichia coli allows tightly controlled. ClyR is a common chimeric lysin that bacteriophage produces to attack target microbes, titratable expression of your protein through the regulation of specific carbon sources such as glucose, glycerol, and arabinose.

TP-I is a cysteine-rich antimicrobial peptide derived from the hemocytes of horseshoe crabs. It is a broad-spectrum antibacterial peptide with good antibacterial function. Team iGEM 2020 SZPT-CHINA test the antibacterial effect of TP-1 on Streptococcus mutans.

We found that previous experiments had shown that ClyR can specifically eliminate Streptococcal microbes which most caries-causing pathogens are. Based on the groups’ contribution, our team design the new composite part BBa_K4030007. We create a gene-engineered E. coli strain containing ClyR gene and a-L-arabinofuranosidase (Arab) gene. Arab is an enzyme used to hydrolyze arabinose from arabinoxylans, a carbohydrate that appears normally in foods. In the strain, ClyR gene is recombined with a promoter that can be induced by arabinose to express ClyR outside of bacteria to function.

Figure 9. The blast results about the amino acid sequence of our new part BBa_K4030007 and the old parts BBa_K3487013..

First of all, we constructed a composite part BBa_K4030007 and transformed it into E.coli. We have successfully obtained the ClyR protein in the culture supernatant of cells carrying plasmids with araB and ClyR genes.

Furthermore, in order to test concentration of araboxylan, we set up different concentration of arabinose to test the fluorescence intensity. The experimental data shows that the optimal concentration of araboxylan is 3%.

In addition, Phage lyase ClyR has a broad bactericidal spectrum, especially the only one reported to be extremely strong against Streptococcus mutans and Streptococcus mulberry. Therefore, our engineering strain has a huge potential commercial market.

Profile

Usage and Biology

Dental caries is a common disease. It not only directly affects human oral health, but also often causes adverse symptoms in other parts of the body. Global disease statistics in 2016 show that the incidence of dental caries in the population is ranking second among common diseases. Studies have shown that the formation of dental plaque is the result of the joint action of a variety of bacteria, including Streptococcus mutans, Lactobacillus, Actinomycetes, etc. Phage lyase ClyR (combined from different bacteriophage lytic enzymes) has a broad bactericidal spectrum, especially the only one reported to be extremely strong against Streptococcus mutans and Streptococcus mulberry. The enzyme is promising to kill these two kinds of streptococci are the main cause of dental caries.

Figure 1. Schematic diagram of ClyR in the prevention or treatment of dental caries..

Construct design

Arab is connected with OpmA and this sequence is inserted into plasmid pUC57_mini (Figure 2 and 3).

Figure 2. J23101-OmpA-arab-TT box..

Figure 3. Schematic map of J23101-OmpA-arab-TT plasmid..

The profiles of every basic part are as follows:

BBa_K4030001

Usage and Biology

BBa_K4030001 is a coding sequence of arab, which is an enzyme used to hydrolyze arabinose from arabinoxylans, a carbohydrate that appears normally in foods.

BBa_K4030000

Usage and Biology

Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane.

Experimental approach

Group 1

Plasmid puc57-kan-mini-J23101-OmpA-araB-TT (Plasmid A) was transformed to E. coli Nissle 1917 by electroporation. We tried to monitor the concentration of the reducing sugar arabinose and thus access the activity of AraB. The concentration of reducing sugar was determined by the DNS kit and the A540 were recorded accordingly.

..

Group 2

Plasmid A and plasmid pBAD-Myc-HisA-OmpA-amilGFP (Plasmid C) were co-transformed to E. coli Nissle 1917 by electroporation. Based on the data of Group 1, the cells containing plasmid A could hydrolyze araboxylan to arabinose. To test the produced arabinose could then induce the expression of genes under the control of pBAD. We choose green fluorescent protein AmilGFP as a reporter protein.

The existence of AmilGFP was monitored by the fluoroscopic examination using SpectraMax i3x..Read the fluoroscopic data of the supernatant, and recorded the average data. After this timepoint, 100 ul of the culture was sampled and marked as “6 h”, “8 h”, “10 h”, “12 h” and “13 h”, respectively, and the fluoroscopic data were recorded accordingly.

Group 3

Plasmid A and plasmid B were co-transformed to E. coli Nissle 1917 by electroporation. The araboxylan with final concentration 0, 0.3%, 0.6%, 1.0%, 1.5% and 2.0% (w/v) was added into the culture to induce ClyR expression.

For the comparison, E. coli Nissle 1917 with plasmid B was cultured by the same way and the expression of ClyR was induced by the addition of arabinose with final concentration of 0 μM, 10 μM,30 μM,0.1 mM, 0.2 mM,0.5 mM and 2 mM.

The protein concentration was monitored at 595 nm using Multiscan Spectrum (BioTek). Read the data for three times, recorded the average of the A595 data.

Gels were scanned with the ImageQuant™ LAS 4000 mini (GE Healthcare).

In vitro activity assay of ClyR

E. coli Nissle 1917 transferred with plasmid A and B, Lactobacillus casei subsp. casei (ATCC334)

1) The culture of Lactobacillus casei subsp. casei (ATCC334)

2) In vitro assay

The value of OD600 was monitored using Multiscan Spectrum (BioTek).

Proof of function

Figure 4. The concentration assay of the reducing sugar in bacteria transformed with plasmid A..

All the data at “0 h” with different concentration of araboxylan was referred as blank, and the concentration of the reducing sugar could be calculated.

As can be seen from figure 4, in the transformed bacteria with plasmid A, the concentration of the reducing sugar increased with the elongation of the incubation time. The plasmid A can work normally and possess hydrolyzing arabinoxylan activity, which supply the arabinose for the expression of ClyR.

AmilGFP expression in cells transformed with plasmid A

Figure 5. The fluoroscopic data of cells transformed with plasmid A..

In the cells transformed with only plasmid A, the fluoroscopic data of cells with the addition of different concentration of araboxylan showed no difference with that of cells without the addition of araboxylan. This data suggested no AmilGFP was produced in cells with or without addition of araboxylan.

AmilGFP expression in cells transformed with plasmids A and C

Figure 6. The fluoroscopic data of cells transformed with plasmids A and C..

In the cells transformed with plasmids A and C, the fluoroscopic data of cells increased with the addition of araboxylan. Meanwhile, the fluoroscopic data become larger with the prolongation of incubation time.

AmilGFP was successfully expressed, suggesting the feasibility and possibility of the inducible secretory expression of ClyR.

..

Figure 7. The SDS-PAGE of supernatant..

The A595 of the culture supernatant of E. coli with plasmid A and B suggested the possible expression of ClyR (Table 2). The A595 data at “0 %” was referred as blank, and the protein concentration could be calculated referred to the standard formula. As can be seen from figure 7, in the transformed bacteria with plasmid A and B, the concentration of the reducing sugar increased with the elongation of the incubation time.

During the expression process, bacteria lysis occurred with the elongation of the incubation time. But the ClyR band could obviously be obtained as is shown in figure 7.

In the engineering cells containing plasmid A and plasmid B, the expression of ClyR was successful.

Figure 8. The in vitro assay of ClyR..

The OD600 of the Lactobacillus casei subsp. casei cell suspension gradually reduced with the addition of supernatant (figure 8).

We have successfully obtained the ClyR protein in the culture supernatant of cells carrying plasmids with araB and clyR genes.

Future plan

We have successfully obtained the AmilGFP expression system, which is induced by arabinose. But we used double plasmid to achieve this aim, which is complicated. In the future, we can adopt a plasmid to realize the expression of aimed gene.