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The pET-21 b (+)_OmpA_DARPin plasmid was then linearized and assembled with the missing DARPin* fragment by In-Fusion. The product was transformed in competent <i>E. coli</i> Stellar cells and transformants were selected on Ampicillin. Plasmids from the resulting colonies were extracted by Miniprep. The presence of the insert was assessed by PCR screening with primers FORWARD: ggttatgctagttattgctcagc and REVERSE: ccgaaacaagcgctcatgagc. Amplification product sizes were checked on EtBr stained agarose gel [data not shown]. Sequence was further validated.
 
The pET-21 b (+)_OmpA_DARPin plasmid was then linearized and assembled with the missing DARPin* fragment by In-Fusion. The product was transformed in competent <i>E. coli</i> Stellar cells and transformants were selected on Ampicillin. Plasmids from the resulting colonies were extracted by Miniprep. The presence of the insert was assessed by PCR screening with primers FORWARD: ggttatgctagttattgctcagc and REVERSE: ccgaaacaagcgctcatgagc. Amplification product sizes were checked on EtBr stained agarose gel [data not shown]. Sequence was further validated.
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The plasmids were finally used to transform <i>E. coli<i/> Tuner cells to hopefully express the DARPin* construction at the cell membrane.
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The plasmids were finally used to transform <i>E. coli</i> Tuner cells to hopefully express the DARPin* construction at the cell membrane.
  
  
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===Functional Parameters===
 
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Latest revision as of 18:40, 10 October 2022


OmpA_DARPin

Gene fusion to express the DARPin fusion protein at the surface of E. coli.

Introduction

This part is composed of the gene coding for the DARPin E2_79 protein (see BBa_K4197019) in fusion with the lpp-ompA-N gene (see BBa_K1694002). This part was designed to link IgE at the surface of E. coli.

The DARPin E2_79 protein has a strong affinity for the constant part of IgE (Baumann et al., 2010). It was merged to the membrane protein Lpp-OmpA-N of E. coli (see BBa_K1694002) to display the DARPin on the surface of E. coli. This lipoprotein is the most abundant in the membrane of E. coli with 100,000 copies per cell (Ortiz-Suarez and al. 2016) and is often used to display proteins on the surface of bacteria (Yang and al. 2016).

Construction

The brick was contructed from the BBa_K4197010 part. To delete the sfGFP fragment, pET-21 b (+)_OmpA_DARPin-sfGFP was linearized by PCR using the high fidelity Phusion DNA polymerase with primers FORWARD: tggtgatgcagtttctgcaaaatttccgcc and REVERSE: gaaactgcatcaccatcaccatcacc, which excluded the sfGFP sequence. The expected size of the amplicon was 6202 bp (Figure 1). Amplification product sizes were checked on EtBr stained agarose gel. Amplicons were further purified from the gel.

Figure 1: pET-21 b (+)_OmpA_DARPin linearized. The expected size of the amplicons was 6202 bp. PCR amplicon sizes were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder is used for the experimental gels. (note that a different ladder is presented on the theoretical gel).
The linearized plasmid was then recircularized by In-Fusion to form the pET-21 b (+)_OmpA_DARPin vector. pET-21 b (+)_OmpA_DARPin was transformed into E. coli Stellar competent cells. Transformants were selected on LB-ampicillin plates. Presence of the insert was checked with a colony PCR using the primers FORWARD: ggttatgctagttattgctcagc and REVERSE: ccgaaacaagcgctcatgagc. Expected size of the fragment were 1162 bp (Figure 2). Expected size of positive colonies was 1885 bp (Figure 2). Plasmids colonies containing the insert were extracted by Miniprep.
Figure 2: identifying the strains that bear pET21 b (+)_Ompa_DARPin by colony PCR. The expected size of the amplicon was 1162 bp. All clones were compared to a control with a linearized plasmid still containing the sfGFP fragment. PCR amplicon sizes were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented on the left and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).

Expression and functionality of the DARPin were assesed, i.e. its capacity to bind IgE. Two experiments were conducted for this purpose, but none of them showed exploitable results (data not shown). As the DARPin protein was HIS-tagged, we first tried to detect it by binding a fluorescent anti-HIS-tag IgG to the cell membrane and measuring fluorescence. No fluorescence was observed, so we could not conclude if the protein was expressed at the membrane, especially without a positive control.

We then tried to assess the functionality of the DARPin by binding an IgE to it (the DARPin being able to link to the constant part of IgE), and then binding a fluorescent anti-IgE IgG to the complex. No fluorescence was observed. Without a positive control, we cannot conclude between an incorrect exposition of the protein (not facing the outer surface), or a mere problem during the setup of the experiment.

The pET-21 b (+)_OmpA_DARPin plasmid was then linearized and assembled with the missing DARPin* fragment by In-Fusion. The product was transformed in competent E. coli Stellar cells and transformants were selected on Ampicillin. Plasmids from the resulting colonies were extracted by Miniprep. The presence of the insert was assessed by PCR screening with primers FORWARD: ggttatgctagttattgctcagc and REVERSE: ccgaaacaagcgctcatgagc. Amplification product sizes were checked on EtBr stained agarose gel [data not shown]. Sequence was further validated. The plasmids were finally used to transform E. coli Tuner cells to hopefully express the DARPin* construction at the cell membrane.

References

More information about the project for which the part was created: DAISY (INSA-UPS 2022)

Other parts of OmpA fusion proteins associated with a DARPin:
- DARPin
- OmpA_DARPin_sfGFP
- OmpA_DARPin fusion + ihfB800-mTagBFP

  1. Baumann, M. J., Eggel, A., Amstutz, P., Stadler, B. M., & Vogel, M. (2010). DARPins against a functional IgE epitope. Immunology Letters, 133(2), 78–84. https://doi.org/10.1016/j.imlet.2010.07.005
  2. Ortiz-Suarez, M. L., Samsudin, F., Piggot, T. J., Bond, P. J., & Khalid, S. (2016). Full-Length OmpA : Structure, Function, and Membrane Interactions Predicted by Molecular Dynamics Simulations. Biophysical Journal, 111(8), 1692–1702. https://doi.org/10.1016/j.bpj.2016.09.009
  3. Yang, Chao; Zhao, Qiao; Liu, Zheng; Li, Qiyun; Qiao, Chuanling; Mulchandani, Ashok; et al. (2016): Cell Surface Display of Functional Macromolecule Fusions on Escherichia coli for Development of an Autofluorescent Whole-Cell Biocatalyst. ACS Publications. Journal contribution. https://doi.org/10.1021/es800441t.s001

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 130
    Illegal NheI site found at 92
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 130
    Illegal BamHI site found at 124
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 130
    Illegal XbaI site found at 47
    Illegal AgeI site found at 832
  • 1000
    COMPATIBLE WITH RFC[1000]