Part:BBa_K4195115

I0500-B0034-LMT sp-his-linker-edl060-B0015

LMT is a signal peptide from the lytic murein transglycosylase of Vibrio natriegens, his-tag enable us to purify the linking protein. LMT here represents a signal peptide used to secrete the protein to the periplasm. Edl060 is a gene encoding endolysin Lysqdvp001 to lyse Vibrio parahaemolyticus. This part is used to verify whether the outer-membrane vesicles can achieve horizontal gene transfer and whether Lysqdvp001 can lyse Vibrio parahaemolyticus. In order to make bond with secondary antibody to detect the produce of Lysqdvp001 as a signal showing successful transformation, a his-tag (6*His) at the N-terminal of Lysqdvp001 was added. And to reduce the possibility that the his-tag may affect the function of Lysqdvp001, a flexible linker between his-tag and Lysqdvp001 was added.

Usage

The circuit is used to verify whether plasmids could be passed from outer-membrane vesicles (OMVs) and whether the plasmids could perform its killing function after transformation. If the transformation was done, the bacteria been transformed will be killed by Lysqdvp001 produced by itself after being induced by L-arabinose. We used both BBa_I0500 and BBa_B0015to construct the expression system and obtained this circuit, which are assembled on the expression vector pUC57-Simple by standard assembly. The constructed plasmids were transformed into E. coli DH5α and BL21(DE3), then the positive transformants were selected by ampicillin and confirmed by colony PCR and sequencing. After a successful construction was confirmed, we carried out outer membrane (OMVs) extraction to obtain OMVs we need to incubate with Vibrio alginolyticus. After incubation and spreading plates with the incubated bacteria culture, we pick up the positive colonies on the plates to do colony PCR to verify if it was transformed correct plasmids. The positive colonies were cultivated and each were divided into two groups. Aliquots of culture were plated out on LB agar plates containing ampicillin to verify the killing effect of Lysqdvp001 on Vibrio alginolyticus. As induced by L-arabinose, experimental group are divided by whether the plate contains L-arabinose or not.

Biology

Lysqdvp001

Lysqdvp001 is an enzyme called endolysin which encoded by gene edl060 from bacteriophage qdvp001 with specificity for Vibrio parahaemolyticus that degrade the peptidoglycan cell wall of Vibrio parahaemolyticus (1).

LMT sp

Lytic murein transglycosylase (LMT) is an enzyme which is able to degrade murein, a component of cell wall of bacteria. There are two kinds of LMTs existing in E. coli: the membrane-binding one and the soluble one. The gene coding LMT homolog is also incorporated in the genome of Vibrio natriegens. The LMT signal peptide (named LMT in our parts) is from the LMT homolog, which can lead the fused protein secreted out of Vibrio natriegens and E. coli.

Characterization

Agarose Gel Electrophoresis

When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target bands(Fig. 1)(2250 bp) at the position between 2000bp and 3000bp.

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195115_pUC57-Simple. Target bands can be observed at the position between 2000 bp and 3000 bp.

OMVs extraction and plasmids packaging verification

The positive colony was cultivated to extract OMVs by hypervesiculation. After extraction, some OMVs were used to PCR to verify if there were target plasmids(Fig. 2) packaging in. The target bands(2250bp) can be observed at the position between 2000bp and 3000bp. And to verify if there's any bacteria remaining in the OMVs extraction, agar spot test was applied.

Fig. 2 Verification of OMV’s ability to load plasmid. a Solid medium plate of agar spot test. b The result of OMV PCR. Contained plasmid is BBa_I0500_pSB1C3.

Fig. 3 Agar spot test of OMVs. b Solid medium plate of agar spot test containing BBa_K4195115.

Incubation

The incubation experiment of OMVs with Vibrio alginolyticus is performed to verify the ability of OMVs enveloping plasmids to transform plasmids. We incubate OMVs with Vibrio alginolyticus for about 12h, and then dilute the incubation product with LB medium added with ampicillin to 102 times and 104 times. The diluted bacteria culture was plated out LB agar plates containing ampicillin. Incubate overnight and observe the colonies on the plate.

Transformation verification

Regular PCR was used to certify the plasmid was correct. We got the target bands(Fig. 3)(2250 bp) at the position between 2000bp and 3000bp.

Fig. 4 DNA gel electrophoresis of the colony PCR products of BBa_K4195115_pUC57-Simple. Target bands can be observed at the position between 2000bp and 3000bp.

Killing effect verification

The positive colonies were cultivated and each were divided into two groups. Aliquots of culture were plated out on LB agar plates containing ampicillin to verify the killing effect of Lysqdvp001 on Vibrio alginolyticus. As induced by arabinose, experimental group are divided by whether the plate contains arabinose or not. After 9h’ cultivation, the result of agar spot test was obserbed and the CFU was counted(Fig. 5).

Fig. 5 The result of killing effect verification. The groups are cultivated from 4 different colonies. Group 1 is cultivated from colony without a correct band so that it serves as control group. Group 2, group 3 and group 4 are colonies with correct bands and serve as experimental groups.

Reference

1. Wang W, Li M, Lin H, Wang J, Mao X. The Vibrio parahaemolyticus-infecting bacteriophage qdvp001: genome sequence and endolysin with a modular structure. Arch Virol. 161(10):2645-52.(2016).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961