Coding

Part:BBa_K2788002

Designed by: chen liang   Group: iGEM18_SZU-China   (2018-10-05)


Encoded by the gene MCL1 from Metarhizium robertsii ARSEF 23

The gene MCL1 from the Metarhizium robertsii ARSEF 23 MCL1 encodes collagen-like protein, which forms a collagenous protective coat to evade insect immune responses.GenBank Acce NO.XM_007819663.1


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 157
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1688
    Illegal NgoMIV site found at 1739
    Illegal NgoMIV site found at 1864
    Illegal AgeI site found at 1670
  • 1000
    COMPATIBLE WITH RFC[1000]

er


iGEM2018 SZU-China

MCL1 from the Metarhizium robertsii ARSEF 23 MCL1 encodes collagen-like protein, which can combine with β-1,3-glucan on the fungus cell wall.β-1,3-glucan is the recognization site for insect hemocytes to recognize and clear incaders.With collagen-like protein,fungus is like putting an "invisible cloak"which can avoid host immune response.Immune-avoidance makes clonization and causing death smoothly. This part was inserted into the pBC expression vector by restriction sites EcoRI and PstI (Fig.1), and the correct construction of this recombinant plasmid was confirmed by PCR identification and sequencing of the PCR products.

Fig.1 Construction of expression vector MCL1-pBC. PgpdA and TtrpC come from parts of 2016_NYMU-Taipei: BBa_K2040101 and BBa_K2040102, and MCL1 comes from the Metarhizium robertsii ARSEF 23. The PgpdA-MCL1-TtrpC part is connected to the pBC plasmid through the BioBrick site.


We transferred the expression vector MCL1-pBC by CaCl ₂ -PEG induction method,then screen transformant by G418 resistance genes and colony PCR.PCR product was identified by agarose gel electrophoresis(Fig.2)

Fig.2 0.8%Agarose Gel Electrophoresis of colony PCR prooduct of the positive clones . The PCR product showed two signal bands at 335 bp and 1817bp respectively, which correspond to the length of M.a primer PCR product and MCL1 primer PCR product. Lane 1: M.a primer PCR product; Lane 2: MCL1 primer PCR product; Lane M: DL marker.


The transformed strain Metarhizium anisopliae 128 was grown in 1/4 SDAY liquid medium, and obtain total RNA by using RNAiso Plus(TAKARA), reverse transcription by using TAKARA PrimeScript™ RT reagent Kit,then perform quantitative PCR.(Fig.3)

Fig.3 quantitative PCR analysis demonstrating WT M.anasopliae and genetically enhanced M.anasopiae mRNA levels,which was increased significantly in transformant.(p<0.05).


We gain the total protein by FastPrep and ultrasonic crushing. The lysate was then centrifuged and the supernatant were electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining(Fig.4)

Fig.4 SDS-PAGE analysis of total protein of wild-type Metarhizium anisopliae 128 and genetically enhanced Metarhizium anisopliae 128. Lane M: Marker Ladder;Lane 1:wild type Metarhizium anisopliae 128;Lane 2 : recombinant Metarhizium anisopliae 128.Lane 2 showed the band corresponded with the molecular weight of collagen-like protein(60.4kDa).


In order to verify the ability of immune-avoidance of M.anasopliae,we inject hyphae homogenate into cockroaches,then extract hemolymph, count the nodules formed of hemocytes(Criteria:more than 10 hemocytes assemble closely)(Fig.5) and observe hemolymph smear under phase contrast microscope(Fig.6)

Fig.5 Change of nodules formed of hemocytes in cockroach hemolymph after injecting hyphae homogenate.At the time point of 0.5h,1h and 8h,the nodules caused by WT M.anasopliae is significantly higher than transformant which means immune-avoidance occurs in genetically enhanced M.anasopliae.(p<0.05)
Fig.6 Immune response of cockroaches’hemocytes to Metarhizium anisopliae spores under phase contrast microscope.Wild-type M.anasoplise triggers more intense immune response than MCL1 transformant, while hemocytes degraded more in transformant groups.
[edit]
Categories
Parameters
None