Part:BBa_K4125001

mlrA +inaK surface display system

2022 Nanjing_NFLS constructed an extracellular enzyme display system to exhibit MlrA enzyme at the surface of E. coli. The InaK protein served as an anchoring motif. This way, the enzyme can directly degrade toxins present in the environment without toxin intake. The genes were transformed on a pET23b plasmid vector.

FIG.1 Schematic of Plasmid, Created with SnapGene

FIG.2 Schematic of Extracellular Enzyme Display

Restriction Endonuclease Digestion

The recombinant plasmid was then extracted to be identified with restriction endonuclease digestion. We separated the targeted gene inaK (537 bp) from the plasmid, and performed 20 rounds of PCR on it. This way, we obtained a larger quantity of the targeted gene, and the gel electrophoresis graph would be of better clarity. The sequence of the primers we used are listed as follows:

Primer	Sequence (5’ to 3’)	Restriction Site
InaK-F	TCGAGCTCCGTCGACAAGCTTATGACTCTCGACAAGGCGTTG	Hind III
InaK-R	TTCCGCATGGTCTGCAAATTCTGCGGC	/
MlrA-F	AATTTGCAGACCATGCGGGAGTTTGTCAAACAG	/
MlrA-R	GTGGTGGTGGTGGTGCTCGAGCGCGTTCGCGCCGGACTT	Xho I
Total-F	AACGGTTTCCCTCTAGAAATAATTT	Hind III
Total-R	CCTTTCGGGCTTTGTTA	Xho I

TABLE 1 Primer sequences and Restriction Sites

e used a 200 bp ladder marker in gel electrophoresis. As shown in FIG.3, there were 3 significant bands respectively around 550 bp, 1000 bp and 1550 bp, each corresponding to inaK, mlrA and combination of the two. This indicated that the plasmid construction had been successful.

FIG.3 Restrictive Endonuclease Digestion Results

Identification of Protein Location

We separated different components of the cell by cell fractionation with ultracentrifuge. The samples of outer membrane, inner membrane and cytoplasm were obtained and stored at -4°C overnight. SDS-PAGE gel electrophoresis was performed the next day. We then used Coomassie Bright Blue to stain the gel and observe the proteins in each sample.

FIG.4 SDS-PAGE Results after Coomassie Staining

The composite InaK + MlrA protein (56 kDa) was found in the outer membrane fraction of pET23b-inaK+mlrA transformed E. coli, verifying our construction. MlrA only (37 kDa) was found in the inner membrane fraction of pET23b-mlrA transformed E. coli, which matched previous literature’s observations. Overall, we verified the compatibility and location of the inaK-based enzyme display system.

Below is our protocol for cell fractionation: 1. The bacteria were grown at 37°C for 12h after 0.1mM IPTG induction.

2. Cells were collected by centrifuge of 6000 rpm for 10 min.

3. After 2 washes with PBS buffer, cells were resuspended in PBS containing 0.1mM EDTA and 10 μg/mL lysozyme.

4. After incubation of 2 hours on ice, the cell suspensions were treated with ultrasound sonication on ice (5 minutes, 1 cycle).

5. Intact cells were separated from the lysates with a slow centrifuge of 6000 rpm for 10 min.

6. At this point, the supernatant contained the whole cell lysates. We retrieved it with ultracentrifugation at 50,000 rpm for 1h to pellet the total membrane fraction.

7. The supernatant containing soluble cytoplasmic and periplasm fractions was collected and stored at -4°C for later assays.

8. The pellet of total membrane fraction was resuspended into PBS buffer containing 0.01 mM MgCl2 and 2% Triton X-100 for solubilizing the inner membrane, and incubated at room temperature for 30 minutes.

9. The suspension was ultracentrifuged again at 50,000 rpm for 1h. The supernatant contained the inner membrane proteins, and the pellet contained the outer membrane proteins.

10. All samples were collected and stored at -4°C for later assays.

FIG.5 Schematic of Cell Fractionation, Created with Biorender

References:

[1]. Li, L., Gyun Kang, D. and Joon Cha, H., 2004. Functional display of foreign protein on surface of Escherichia coli using N‐terminal domain of ice nucleation protein. Biotechnology and bioengineering, 85(2), pp.214-221.

[2]. Shi, H. and Su, W.W., 2001. Display of green fluorescent protein on Escherichia coli cell surface. Enzyme and microbial technology, 28(1), pp.25-34.

[3]. Liu, M., Ni, H., Yang, L., Chen, G., Yan, X., Leng, X., Liu, P. and Li, X., 2019. Pretreatment of swine manure containing β-lactam antibiotics with whole-cell biocatalyst to improve biogas production. Journal of Cleaner Production, 240, p.118070.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
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• 23
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• 25
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  Illegal NgoMIV site found at 1251
  Illegal AgeI site found at 429
  Illegal AgeI site found at 786
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 238