Difference between revisions of "Part:BBa K2971000"
 
(5 intermediate revisions by the same user not shown)
Line 12: Line 12:
 
<i>Escherichia coli</i>, by inserting it into a vector along with the genes <i>crtB</i> and <i>crtI</i>. This composite part produced red cells when expressed in Escherichia coli confirming that the part functions as intended.  
 
<i>Escherichia coli</i>, by inserting it into a vector along with the genes <i>crtB</i> and <i>crtI</i>. This composite part produced red cells when expressed in Escherichia coli confirming that the part functions as intended.  
  
−
The part was cloned into an expression vector under the arabionose inducible promotor <i>araC</i>. Successful cloning was confirmed with colony PCR of transformed colonies (figure 1). Colonies that carried the insert was sequenced to check for the potential presence of mutations or frameshift.  
+
The part was cloned into an expression vector under the arabionose inducible promotor <i>araC</i>. Successful cloning was confirmed with colony PCR of transformed colonies (figure 1). Transformed colonies carrying the insert would have a shifted band relative to the control. Colonies that carried the insert was sequenced to check for the potential presence of mutations or frame-shifts.
 +
 
 +
<html>
 +
<img style="width:40% !important;" src="https://2019.igem.org/wiki/images/9/91/T--UiOslo_Norway--basicColonyPCR.png">
 +
<p>
 +
<strong>Figure 1: Colony-PCR of cells transformed with our basic parts (crtE, crtI and crtB)</strong></br>
 +
Primers were designed to amplify the part of the intended plasmid that contains the insert.</br>
 +
Numbers denote the colony checked with the primers. 14 and 23 are PCR products from empty vectors that function as controls. Colony 1-12 and 15 shows that we had several colonies of crtE with the insert. 16-20 shows colonies carrying the crtB insert. 21 show that we had one colony with the insert.  
 +
</p>
 +
</html>
 +
 
  
 
We expressed the protein in <i>E. coli</i> (DH5&#945;)(figure 2). We detected a faint protein band during our SDS analysis; it is likely the expression was not well-optimized.  
 
We expressed the protein in <i>E. coli</i> (DH5&#945;)(figure 2). We detected a faint protein band during our SDS analysis; it is likely the expression was not well-optimized.  
  
 +
 +
<html>
 +
<img style="width:40% !important;" src="https://2019.igem.org/wiki/images/0/07/T--UiOslo_Norway--SDS.png">
 +
<p>
 +
<strong>Figure 2: SDS-page of colonies expressing our basic parts</strong></br>
 +
The expression of our basic parts were tested in E.coli (DH5&#945;). We could detect a faint band of CrtE and CrtI.
 +
</p>
 +
</html>
  
 
'''References'''
 
'''References'''

Latest revision as of 00:07, 22 October 2019


crtE from Deinococcus radiodurans

This part is the gene crtE (dr1395) from the extremophile Deinococcus radiodurans and encodes geranylgeranyl diphosphate synthase (GGPPS). GGPPS catalyze the condensation of farnesyl pyrophosphate (FPP) and isopentenyl pyrophosphate (IPP) into geranylgeranyl diphosphate (GGPP)[1]. GGPP is a precursor for the carotenoid biosynthetic pathway. The sequence has been codon optimized for expression in Escherichia coli and iGEM registry compatibility.

The UiOslo 2019 team used this gene in a composite part (BBa_K2971004) to produce the red pigment lycopene, in Escherichia coli, by inserting it into a vector along with the genes crtB and crtI. This composite part produced red cells when expressed in Escherichia coli confirming that the part functions as intended.

The part was cloned into an expression vector under the arabionose inducible promotor araC. Successful cloning was confirmed with colony PCR of transformed colonies (figure 1). Transformed colonies carrying the insert would have a shifted band relative to the control. Colonies that carried the insert was sequenced to check for the potential presence of mutations or frame-shifts.

Figure 1: Colony-PCR of cells transformed with our basic parts (crtE, crtI and crtB)
Primers were designed to amplify the part of the intended plasmid that contains the insert.
Numbers denote the colony checked with the primers. 14 and 23 are PCR products from empty vectors that function as controls. Colony 1-12 and 15 shows that we had several colonies of crtE with the insert. 16-20 shows colonies carrying the crtB insert. 21 show that we had one colony with the insert.


We expressed the protein in E. coli (DH5α)(figure 2). We detected a faint protein band during our SDS analysis; it is likely the expression was not well-optimized.


Figure 2: SDS-page of colonies expressing our basic parts
The expression of our basic parts were tested in E.coli (DH5α). We could detect a faint band of CrtE and CrtI.

References

1. Liu, C., Sun, Z., Shen, S., Lin, L., Li, T., Tian, B., & Hua, Y. (2014). Identification and characterization of the geranylgeranyl diphosphate synthase in Deinococcus radiodurans. Letters in Applied Microbiology, 58(3), 219-224. doi:10.1111/lam.12181



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 841
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 43