Part:BBa_K1413001:Design

P0 promoter-RBS B0032-sfGFP- Terminator B0015 - Pr promoter-DmpR

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1241
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1719
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1404
  Illegal BsaI.rc site found at 1945
  Illegal SapI.rc site found at 211
  Illegal SapI.rc site found at 2602
  

Design Notes

We performed an assembly of the two parts by :
-digesting BBa_K1031222 with EcorI and XbaI while digesting BBa_K1031211 by EcorI and SpeI.
-ligating the two digestion products.

Figure 1 : Gel electrophoresis of plasmid extracted from 4 colonies of DH5alpha transformed with ligation product
This gel uses a 2-log DNA ladder.

BBa_K1031211 (Dmpr) had an extra Pst1 restriction site at position 226 (position 0 being EcoR1 restriction site) (figure 2). Thus when digesting by PstI we obtained two fragment of 2252bp and 1584bp (the entire plasmid measuring 3836bp.

Figure 2 :Gel electrophoresis of pstI digested plasmids extracted from 4 colonies of DH5alpha containing BBa_K1031211(DmpR)

We ordered several sample of this part but all of them were carrying two Pst1 site.
We thus performed several trials of site directed mutagenesis and finally managed to modify a single nucleotide in the Pst1 site, with respect to the amino acids sequence.
We used the two following primers :
FW: CGCATGCTGTTGCTTCAGTTTTCAGCGATGGC
RV: GCCATCGCTGAAAACTGAAGCAACAGCATGCG
We performed a PCR with one tube containing the Forward primer and a second one with the Reverse primer :
-1µL Primer
-1µL BBa_K1413001 plasmid
-1µL Dntp
-0.5µL Q5 High Fidelity
-10µL Q5 buffer
-35.5µL H20
Each tubes will carry double strand plasmids and one of the strands is containing the mutated sequence. After this PCR run both PCR tubes are mixed and the resulted mix is placed at 95°C for 1 min. This allows denaturation of double strand DNA.
When the temperature cools down, DNA re-anneal which generate double strand DNA with both strand containing the mutated sequence.
Then the sample is treated with DpnI during 2h at 37°C allowing digestion of methylated and hemimethylated DNA (methylation being only present on parental DNA that does not contain the mutation)
Thus only non methylated double strand plasmid with mutated sequence is present in the tubes.
DH5alpha competent cells were then transformed by heatshock. Figure 3 shows that mutation has been succesfull on the ligation product of BBa_K1031211(DmpR) and BBa_K1031222 (sfGFP).
.

Figure 3 :Gel electrophoresis of digested plasmids extracted from 4 colonies of DH5alpha containing BBa_K1031211(DmpR) and BBa_K1031222 (sfGFP) ligation product
1= digestion by EcoRI; 2= digestion by PstI; 3= double digestion by EcoRI and PstI

===Source=== This part is a composition of two parts from Peking iGEM 2013: BBa_K1031222 (sfGFP) & BBa_K1031211 (DmpR) Professor V.Shringler provided 2013 Peking iGEM Team with pVI401 composed of Pr-DmpR on vector pVI39. ===References===