Part:BBa_K1894001

shuttle plasmid which can replicate in cyanobacteria and E.coli

This part is originally a self-constructed shuttle plasmid that can both replicate in cyanobacteria and E.coli, but we extract its key sequence and link it to pSB1C3 plasmid backbone. The shuttle plasmid we constructed possesses the replication origins of E.coli plasmid and cyanobacterium plasmid, CaMV35S promoter, multiple cloning sites (MCS) and rbcS polyA terminator, Which makes it convenient to insert and express target gene and to screen out the recombinants. Ori site of cyanobacterium plasmid comes from indigenous plasmids pPbs extracted from Plectonema boryanum.

Usage and Biology

After we recover gene segment of GvpA1 from cloning plasmid pET30a(+), we then link GvpA1 gene with shuttle plasmid pPKE2. We introduce shuttle plasmid into our experiments because we need to transform both E.coli and microcystis aeruginosa during experiments. （The plasmid of microcystis aeruginosa itself does not contain selectable marker and cannot replicate in E.coli. Traditional plasmid vector used to transform E.coli cannot transform microcystis either. ）The shuttle plasmid pPKE2 we constructed and used possesses the replication origins of E.coli plasmid and cyanobacterium plasmid, CaMV35S promoter, multiple cloning sites (MCS) and rbcS polyA terminator subcloned from plasmid pKYLX一71.35, which makes it convenient to insert and express target gene and to screen out the recombinants. Ori site of cyanobacterium plasmid comes from indigenous plasmids pPbs extracted from Plectonema boryanum.

Characterization of the part BBa K1894001

The goal of the experiment is to prove that the complete shuttle plasmid we constructed can both replicate in E.coli and cyanobacteria. Therefore, three measures were taken, using methods of transformation, resistance screening and endonuclease identification.

• Use shuttle plasmid to transform E.coli.
• Positive clones were screened by kanamycin and plasmid DNA extracted is identified with restriction endonuclease.
• Conduct basic kanamycin resistance test of microcysis.
• Use shuttle plasmid to transform microcysis.
• Carry out kanamycin resistance screening after the shuttle plasmid was transformed into microcysis.

Considering that the shuttle plasmid contains kanamycin gene, we decided to carry out kanamycin resistance screening. Basic kanamycin resistance test of Microcystis aeruginosa shows that the algae cannot resist concentration of 5μg/mL and above on BG11. Therefore, we choose 10-15μg/mL kanamycin for screening. After shuttle plasmid is transformed into microcysis, transposon screening is carried out by 7-10 days of culturing on BG-11 medium which contains 15µg/mL of kanamycin.

First experiment: Validation of E.coli Transformation

After transformation of E.coli BL21(DE3), we successfully screened out the positive clones and extracted the recombinant plasmid (the shuttle plasmid) containing GvpA1 gene for restriction endonucleases identification.

Figure 1: Electropherogram of plasmid pPKE2 DNA

A1:products after EcoRI digestion
A2: products after BamHI digestion
M: λDNA marker for Hind III digestion
B1: DNA of recombinant plasmid after EcoRI and SphI digestion
B2: Recombinant plasmid pPKE2
M: λDNA marker for EcRI and HindIII digestion

The plasmid should have a length of 9.8kb after ligation with target gene GvpA1, which is verified and showed in A1. Gene segment of GvpA1 and pPKE2 both include BamHI sites and two separated segments with length 2.8kb and 7.0kb each will show up after BamHI digestion， which is verified in A2. When gene segment of GvpA1 is inserted into the plasmid, a new SphI site will show up between original EcRI-SphI segment (2.8kb). Therefore, two separated segments with length 1.3kb and 1,5kb each will show up after EcoRI and SphI digestion, which is verified in B1.

Second experiment: Validation of Microcystis transformation

Fig.2. growth condition of microcystis a week after transformation

Left: control group (no recombinant plasmid introduced)
Right: Microcystis of Recombination group appears on BG11 medium containing kanamycin

Assembly Compatibility:

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

  Illegal NheI site found at 5892
  Illegal NotI site found at 6620
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 6613
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1596
  Illegal NgoMIV site found at 1756
  Illegal NgoMIV site found at 4803
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2676