Difference between revisions of "Part:BBa K2779912"
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We wanted an araBAD-based expression system because:  
 
We wanted an araBAD-based expression system because:  
  
−
It is inducible by L-arabinose and is tightly regulated by araC;
+
#It is inducible by L-arabinose and is tightly regulated by araC;
−
It consists of a strong promoter  for increased expression;
+
#It consists of a strong promoter  for increased expression;
−
It is compatible with our preferred workhorse strain, DH10B, allowing us to perform our cloning and expression work in the same strain; and
+
#It is compatible with our preferred workhorse strain, DH10B, allowing us to perform our cloning and expression work in the same strain; and
−
It is more cost-effective, since L-arabinose is more economical than IPTG.
+
#It is more cost-effective, since L-arabinose is more economical than IPTG.
−
When we initially looked through the repository, we found that while <a href="https://parts.igem.org/Part:BBa_K1602055"> BBa_K1602055</a>"), which had GFP under the control of araC/pBAD, was the closest to our needs, there was no easy way for us to replace their inserted GFP with our desired genes, and there was no way for us to collect the expressed enzymes. Thus, we decided to improve BBa_K1602055.
+
  
 +
When we initially looked through the repository, we found that while BBa_K1602055 (for Parts Page: https://parts.igem.org/Part:BBa_K1602055), which had GFP under the control of araC/pBAD, was the closest to our needs, there was no easy way for us to replace their inserted GFP with our desired genes, and there was no way for us to collect the expressed enzymes. Thus, we decided to improve BBa_K1602055.
 +
 +
'''The translation of the 6xHis tag occurs in the +2 frame. This means that to ensure proper fusion of the 6xHis tag to your protein of interest, users must include one spacer nucleotide between the restriction enzyme recognition site and the first codon of the gene of interest.'''
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 +
For a full description of all the differences between this construct, which is based out of Invitrogen’s pBAD/His expression vector, and BBa_K1602055, as well as the rationale behind these modifications, please proceed to our Wiki.  Briefly,  there were three significant modifications from BBa_K1602055. The most critical modification is the inclusion of a 6xHis tag in the N-terminus. Another significant modification is the addition of a BamHI and XhoI site upstream of the GFP. Lastly, the GFP variant used in this construct contains the A206K mutation, which makes this GFP variant an obligate monomer. 
 +
 +
'''The translation of the 6xHis tag occurs in the +2 frame.''' This means that to ensure proper fusion of the 6xHis tag to your protein of interest, users must '''include one spacer nucleotide''' between the restriction enzyme recognition site and the first codon of the gene of interest.
 +
<br>
 +
 +
'''Protein Purification Experiment'''
  
 
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Revision as of 18:34, 17 October 2018


L-arabinose-induced eGFP with 6xHis tag

Team UAlberta’s cloning efforts to clone our protoporphyrin IX biosynthesis pathway relied heavily on the use of visible and fluorescent markers to assemble our desired constructs. In order to characterize our enzymes, we wanted a system that would enable us to easily clone, express and purify each of our enzymes for further experimentation.

We wanted an araBAD-based expression system because:

  1. It is inducible by L-arabinose and is tightly regulated by araC;
  2. It consists of a strong promoter for increased expression;
  3. It is compatible with our preferred workhorse strain, DH10B, allowing us to perform our cloning and expression work in the same strain; and
  4. It is more cost-effective, since L-arabinose is more economical than IPTG.

When we initially looked through the repository, we found that while BBa_K1602055 (for Parts Page: https://parts.igem.org/Part:BBa_K1602055), which had GFP under the control of araC/pBAD, was the closest to our needs, there was no easy way for us to replace their inserted GFP with our desired genes, and there was no way for us to collect the expressed enzymes. Thus, we decided to improve BBa_K1602055.

The translation of the 6xHis tag occurs in the +2 frame. This means that to ensure proper fusion of the 6xHis tag to your protein of interest, users must include one spacer nucleotide between the restriction enzyme recognition site and the first codon of the gene of interest.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1476
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1346
    Illegal BamHI site found at 1532
    Illegal XhoI site found at 1549
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1181
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1398
    Illegal SapI site found at 1163