Coding sequence for Tar-EnvZ fusion protein. Tar is the bacterial chemotaxis receptor for aspartate. EnvZ is an osmosensor kinase, which, through phosphorylation of OmpR, regulates transcription of porin genes.
This fusion protein combines the extracellular and transmembrane domains of Tar with the intracellular domain of EnvZ. The fusion converts chemical binding of aspartate to production of transcription factors.
Usage and Biology
Tar-EnvZ consists of 484 amino acid residues. The NH2 terminal has 255 residues derived from Tar, and the COOH terminal has 229 residues derived from EnvZ. The two genes are fused at their unique NdeI (CATATG) restriction sites, as described in Utsumi R., et al (1989). <br/>P07017 MCP-II Aspartate Receptor Protein <br/>P02933 ENVZ_ECOLI
2016, the iGEM Team Hamburg (Finding Chlamydori) worked on and modifying the receptor tar-envZ. This receptor is registered in the iGEM data bank as a fusion protein of the tar-domain and the intracellular EnvZ-domain, although the receptor has an aspartate-binding domain consisting of 484 amino acids. 255 amino acids belong to the N-terminal Tar-domain and 229 amino acids to the C-terminal EnvZ-domain. The assembled construct is 1516 base pairs long and registered as BBa_C0082. from the iGEM Team Antiquity. The function of both domains has already been thoroughly investigated Utsumi et al.  und Mise et al. , thereby only the periplasmic aspartate-binding domain (amino acids 26 to 193) has been expressed and studied. In the original paper by Mise et al. , the Tar-domain was amplified out of the genome of Escherichia coli DH5a with primer pair 5‘ – ATG GCT AGC GAT GAC GAC GAC AAG GGC AGC CTG TTT TTT TCT TC – 3‘ (forward) and 5‘ – CTC GAA TTC TCA TTA TTG CCA CTG GGC AAA TC – 3‘ (reverse), and cloned in the expression vector pET-28a(+). In addition to the N-terminal start methionine, the vector has a thrombine cleavage site upstream of the C-terminal his-tag. According to the paper, tar domain was purified using the His-tag. His-tag renders the protein aggregation-prone, thus it has been removed afterwards through the thrombine cleavage site. The non-tagged protein was used for a crystallography. The team last year did not investigate this part further. Instead, the Tar-EnvZ-receptor was cloned in an OmpR/GFP reporter construct and the GFP concentration measured after a mDAP induction of cells that were transformed with the whole construct. The results were published (http://2016.igem.org/Team:Hamburg/), although the yields might have been higher, if the aggregation propensity has been known before. Our goal this year was to investigate whether the his-tagged tar-protein aggregate completely or only to a certain degree and how this can be prevented. We used the expression vector pET-28a(+) with N-terminal methionine and C-terminal His-tag for both the Tar-domain from Mise et al. and the biobrick BBa_C0082. Instead of only using the sequence 26 to 193 amino acids, we used the entire 255 amino acid sequence of the tar-receptor of the Tar-EnvZ-receptor. We used the following primers: Forward primer: 5'-ATT GCA GCT AGC ATG ATT AAC CGT ATC CGC GTA GTC ACG CTG TTG G-3' Reverse primer: 5'-ATT GCA GGA TCC TGA AAC GCT CTG CGC CAG GTC GC-3' The product is 765 base pairs long (255 amino acids) and has a molecular weight of 31.2 kDa. With this primer pair and a DNA sample of the biobrick BBa_C0082 provided by the iGEM Headquarters we amplified the domain using the flowing PCR reaction: 1. 10x Pfu Polymerase Buffer 5.0 µL 2. dNTP’s (10 mM each) 1.0 µL 3. Forward Primer 1.25 µL 4. Reverse Primer 1.25 µL 5. BBa_C0082 (25 ng/µL) 1.0 µL 6. Pfu Polymerase (2.5 U/µL) 1.0 µL 7. Distilled Water 39.5 µL Total volume 50.0 µL PCR program: 1. Initial Denaturation 98 °C 2 min 2. A. Denaturation 98 °C 30 sec B. Annealing 65 °C 30 sec C. Elongation 72 °C 1 min GO TO A -> repeat 25x 3. Final Extension 72 °C 10 min
The PCR product was purified using the GeneJET PCR Purification Kit by Thermo Fischer Scientific and eluded in 20 µL water. The purified product was cut with the NEB enzymes NheI und BamHI: 1. BamHI-HF 1.0 µL 2. NheI-HF 1.0 µL 3. CutSmart Buffer 5.0 µL 4. PCR Product 20.0 µL 5. Distilled Water 23.0 µL Total volume 50.0 µL
The pET-28a(+) vector was restricted with both enzymes, but instead of adding the entire 20 µL PCR product only 10 µL vector were used and 10 µL more water. Both samples were incubated at 37 °C for one hour and purified with the Thermo Fischer Scientific PCR Purifikation Kit. Therafter, the vector was dephosphorylated: 1. Restricted and purified Plasmid 10.0 µL 2. 10X reaction buffer for AP used in reaction 2.0 µL 3. FastAP Thermosensitive Alkaline Phosphatase 1.0 µL (1 U) 4. Distilled water 7.0 µL Total volume 50.0 µL
The dephosphorylation was incubated at 37 °C for 30 minutes and heated to 75 °C for 5 minutes to stop the reaction. Afterwards vector and insert were ligated with the Thermo Fischer Scientific T4 ligase. 1. Linear vector DNA 100 ng 2. Insert DNA 1:5 molar ration over vector 3. 10x T4 DNA Ligase buffer 2.0 µL 4. T4 DNA Ligase 1.0 µL (1 U) 5. Destilled water up to 20 µL Total volume 20.0 µL
The ligation mixture was transformed into E.coli DH5a cells (following the protocol from Zang Gong ). Four colonies picked and the plasmid was isolated with the GeneJET Plasmid Miniprep Kit by Thermo Fischer Scientific and verified by sequencing. A colony-PCR was not performed, since the tar-receptor can be found in the native E. coli genome and therefore every colony would be positive. A plasmid isolated from one colony of E.coli DH5a cells (verified by sequencing) was transformed and expressed in E.coli BL21(DE3). Therefore, 50 µL kanamycin [50 mg/mL] and 500 µL of the pET-28a(+) / Tar pre-culture were added to 50 mL fresh LB-media in a 300 mL flask and incubated at 37 °C at 220 rpm. The OD at 595 nm was measured every half hour and a 1 mL sample taken at a OD [595 nm] = 0.3. The culture was divided into 20 mL cultures and transferred in 100 mL flasks. One of the flasks was induced with 20 µL 400 mM IPTG and further incubated for 2h. The OD [595 nm] was measured every 30 min and a 1 mL sample for analysis was withdrawn every hour. The samples were centrifuged at 4 °C and 12,000 g for 10 minutes, the media was removed and cell pellets stored at -20 °C.
Table 1: Growth curve of the expression test of pET-28a(+): tar in E.coli BL21(DE3) at 37 °C, 220 rpm and OD at 595 nm.
Figure 1: OD [595 nm] as a time course during growth of the pET-28a(+)/Tar expression test at 37 °C und 220 rpm. The culture was divided after 145 min and one was induced with IPTG and incubated for two additional hours. The expression was analysed on SDS polyacrylamide electrophoresis (SDS-PAGE). • Stacking gel (5 %) 30 % Polyacrylamide 0.85 mL H20 3.40 mL 1 M Tris-HCl (pH 6.8) 0,625 mL 10 % APS 50 µL 10 % SDS 50 µL TEMED 5 µL ______________________________________ Total volume 5 mL
• Separating gel (12 %) 30 % Polyacrilamide 4 mL H2O 3,3 mL 1.5 M Tris-HCL (pH 8.8) 2,5 mL 10 % APS 100 µL 10 % SDS 100 µL TEMED 5 µL _______________________________________ Total volume 10 mL
The SDS-PAGE gels were prepared and stored at 4 °C in a wet cloth after polymerisation. Cell pellets were mixed with 2x SDS - loading buffer containing dye and ß-mercaptoethanol (for equal dilution of the samples the following was considered: OD[595 nm] = 0,1 was mixed with 100 µL of 2x SDS-loading buffer). The samples heated to 95 °C for 5 minutes and cooled down on ice. Two parallel 12% SDS-PAGE gels were loaded with samples. One gel was used for Coomassie staining and one for immunostaining. The gels were run at 10 mA for the stacking gel and 60 mA for the separating gel. The Coomassie stain was put in a Coomassie-Neuhaus/methanol solution and destained with water. The gel for the immunoblot was blotted on a PVDF membrane, blocked in 5 % milk powder in TBST buffer, and subsequently washed in TBST-buffer. Thereafter, the membrane was incubated with the first antibody, a mouse anti-His-antibody (dilutions 1:XXX), washed in TBST-buffer and incubated with the second goat-anti-mouse-HRP-antibody in TBST (dilution 1:XXX) containing 1 % milk powder. The detection was performed with ECL – solution (solution I: 100 µL luminole, 44 µL cumarine acid, 1 M Tris/HCl pH 8.5 with 8,85 mL water and solution II: 6 µL 30% H2O2, 1 mL 1 M Tris/HCl pH 8.5 with 9 mL water).
Figure 2: Coomassie stained SDS-PAGE. Gel loading scheme: lane 1 unstained protein ladder (Thermo Fisher Scientific), lane S - cell pellet before the culture was split; lane 1h/C - cells of the non-induced flask after one hour; lane 1h/I – cells induced with IPTG for 1h; lane 2h/C – cells from non-induced culture at 2h; lane 2h/I – cells induced with IPTG for 2h. Cell pellets were dissolved in SDS-loading buffer as described above and in each lane 20 µl were loaded.
Figure 3: Immunoblot of the same gel (Fig. 2) detected with anti-His-antibody. Loading scheme: lane 1 prestained protein ladder (Thermo Fisher Scientific), lane S - cells pellet before the culture was split; lane 1h/- - cells of the non-induced flask after one hour; lane 1h/+ – cells induced with IPTG for 1h; lane 2h/- – cells from non-induced culture at 2h; lane 2h/+ – cells induced with IPTG for 2h.
Two bands were detected - a prominent one at about 30 kDa and a thinner one at about 32 kDa).
In the Coomassie-gel the band of the tar-protein was not detectable, whereas it was clearly visible on the immunoblot and as expected only in the induced samples. Thus, we proved the expression of the protein with non-cleaved His-tag. The expression was repeated in lager expression volume (2.5 L) to investigate whether the protein can be purified without cleaving the His-tag, which is suggested to trigger partitioning of the protein into aggregates. The cells were cultured to an OD [595 nm] of 0,8, induced for 1h and collected by centrifugation. In the following, the samples were stored overnight at 4°C and resolved in NPI-10 buffer next morning. One sample was subjected to lysis by lysozyme and the other was lysed by mechanical treatment with Retsch-mill. His-tagged protein was purified with nickel-NTA-column and fractions from the washing and elution step were collected. The first SDS-PAGE gels that were made could not be used for an evaluation since there are no separate lanes. For a better result, the first fraction of the washing step of the Redschmill-sample and the first three fractions of the elution were loaded on another SDS-PAA gel.
Figure 4: 12% SDS-PAGE gel stained with Coomassie to evaluate the purification. Gel loading scheme: lane 1- prestained protein ladder (Thermo Fisher Scientific), lanes 2-4 - first three fractions of the eluate of cells lysed with lysozyme; lane 5 - fraction after the first washing step of cells lysed with the Retsch-mill; lanes 6-8 - first three fractions of the eluate of cells lysed lysed with the Retsch-mill; 20 µL were loaded in each lane.
Clearly, the purification using His-tag was successful which is evidenced by the most intensive band at 30 kDa (lane 2-5 and 6-8, Fig. 4). Notably, there is no band in the wash fraction. The intensity of the band decreases with the increased elution volume (e.g. from lane 2 to 5 and form lane 6 to 8, Fig. 4) suggesting that the protein was quickly eluted with the first volume of the elution buffer. The successful purification is a clear proof that it is possible to purify this protein using the His-tag which does not seem to cause any aggregation. The latter might be due to the fact that we express short and might be a problem at long induction cycles.
 Utsumi, R., Brissette, R. E., Rampersand, A., Forst, S. A., Oosawa, K., Inonye, M. (1989). Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate  Mise, T., Matsunami, H., Samatey, F. A., Maruyama, I. N. (2014). Crystallization and preliminary X-ray diffraction analysis of the periplasmic domain of the Escherichia coli aspartate recept0r Tar and its complex with aspartate
Sequence and Features
- 10COMPATIBLE WITH RFC
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- 1000INCOMPATIBLE WITH RFCIllegal SapI.rc site found at 111