Difference between revisions of "Part:BBa K1898250"
Line 24: Line 24:
 
===Sequencing===
 
===Sequencing===
 
We set up PCR to check if the DNA have the expected bands. We saw the bands at their expected size, which is ~2.1kb.  
 
We set up PCR to check if the DNA have the expected bands. We saw the bands at their expected size, which is ~2.1kb.  
−
The DNA was then sent to sequencing. The sequencing results are unable to confirm the sequence from 854 to 895 bp. The four cutting sites are highlighted in red, promoter + rbs highlighted in light blue, GSR in orange, 10x Histidine-Tag in green, and terminator in dark blue.  
+
The DNA was then sent to sequencing. The sequencing results are unable to confirm the sequence from 854 to 895 bp.  
 +
*The four cutting sites are highlighted in red
 +
*BBa_K880005 is highlighted in light blue
 +
*GSR is highlighted in orange
 +
*10x Histidine-Tag in highlighted in green
 +
*BBa_B0015 is highlighted in dark blue.  
  
 
Sequence with vf2 primer:
 
Sequence with vf2 primer:
Line 32: Line 37:
 
Sequence with vr primer:
 
Sequence with vr primer:
  
−
https://static.igem.org/mediawiki/parts/7/7f/Bght_vf2.png
+
https://static.igem.org/mediawiki/parts/0/0c/Bght_vr.png
  
 
Although 854 to 895 bp is not confirmed in the two files provided here, this GSR DNA comes from BBa_K880005. From the sequencing file uploaded (https://parts.igem.org/Part:BBa_K1898200), 854 to 895 bp of the GSR gene is confirmed to be correct.
 
Although 854 to 895 bp is not confirmed in the two files provided here, this GSR DNA comes from BBa_K880005. From the sequencing file uploaded (https://parts.igem.org/Part:BBa_K1898200), 854 to 895 bp of the GSR gene is confirmed to be correct.

Revision as of 15:51, 18 October 2016


Strong promoter + Strong RBS + GSR + 10x Histidine tag + Double terminator

BBa_K880005 consists of a strong promoter and strong RBS and was used to maximize protein production. This construct codes for GSR (glutathione reductase) and a 10x Histidine-tag. GSR is a catalyst for the conversion of glutathione disulfide to glutathione and 10x Histidine-tag is used for protein purification. BBa__B0015 is a double terminator used to stop transcription.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 804
    Illegal BamHI site found at 1488
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 89
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1199
    Illegal SapI.rc site found at 1607



Sequencing

We set up PCR to check if the DNA have the expected bands. We saw the bands at their expected size, which is ~2.1kb. The DNA was then sent to sequencing. The sequencing results are unable to confirm the sequence from 854 to 895 bp.

  • The four cutting sites are highlighted in red
  • BBa_K880005 is highlighted in light blue
  • GSR is highlighted in orange
  • 10x Histidine-Tag in highlighted in green
  • BBa_B0015 is highlighted in dark blue.

Sequence with vf2 primer:

Bght_vf2.png

Sequence with vr primer:

Bght_vr.png

Although 854 to 895 bp is not confirmed in the two files provided here, this GSR DNA comes from BBa_K880005. From the sequencing file uploaded (https://parts.igem.org/Part:BBa_K1898200), 854 to 895 bp of the GSR gene is confirmed to be correct.