Difference between revisions of "Part:BBa K1668003"

 
 
(13 intermediate revisions by 2 users not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1668003 short</partinfo>
 
<partinfo>BBa_K1668003 short</partinfo>
  
−
metK is the S-adenosylmethionine synthetase gene from Streptomyces avermitilis, which was found to stimulate the production of avermectins. When wild-type strain ATCC31267 was transformed with pYJ02 and pYJ03, two metK expression plasmids, avermectin production was increased about 2.0-fold and 5.5-fold compared with that in the control strains, respectively. The avermectin productivity of each culture was quantitatively measured by HPLC analysis.  
+
<br>
 +
The part orfX is a putative membrane-bound putative regulatory gene and its product is a putative membrane protein.
 +
<br>
 +
<br>
 +
This gene sequence could not function in E.coli. If you would like to express frr in Streptomyces avermitilis, remember to add ermEp [https://parts.igem.org/Part:BBa_K1668004] as its promoter.
  
−
As the kinetic study revealed, avermectin overproduction in recombinant strain was not caused by the change of cell growth rate or copy number effect. Instead, metK stimulates the avermectin production by increasing the intracellular concentration of S-adenosylmethionine (SAM), an important intermediate product in avermectin production. However, there may be a maximum concentration of SAM for the production of avermectin in S. avermitilis, over which it has no any effect on the antibiotic production.
+
<!-- -->
  
−
The results of experiments showed that there were different effects of metK expression levels on avermectin production in various S. avermitilis strains. The gene expression levels of metK in two engineered strain, GB-165 and 76-05, were much higher then those in wild-type strain, whereas the avermectin productivity in these two strains were not significantly improved. It probably because the high expression level of metK limited the increasement of avermectin by overexpression of metK.
 
−
 
−
 
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
−
 
−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1668003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1668003 SequenceAndFeatures</partinfo>
Line 22: Line 18:
 
<partinfo>BBa_K1668003 parameters</partinfo>
 
<partinfo>BBa_K1668003 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
<h2>'''Characterization'''</h2>
 +
<h3> BACKGROUND </h3>
 +
orfX is a putative membrane-bound putative regulatory gene and its product is a putative membrane protein.
 +
<h4>Function</h4>
 +
The results of PCR analysis and the gene disruption experiments strongly suggest that either a considerably conserved sequence of orfX or a combination of orfX and other assisting genes exists in the high producers. And the orfX product appears to play an essential role in the production and regulation of avermectin in both the normal strain and the high producers. When wild-type S. avermitilis was transformed with a 8.0-kb DNA fragment containing the orfX gene, avermectin production increased approximately 3.5-fold. However, the nature of the stimulatory effect of orfX is still unclear.
 +
<h4>Principle</h4>
 +
The orfX gene reveals a “copy number effect”. That is to say, multiple fragment copies can substantially increase avermectin production in S. avermitilis. Different from metK and frr gene, the DNA fragment containing orfX gene also increased avermectin bio-synthesis in various S. avermitilis strains, including the high-producing mutant strain ATCC 31780 and a semi-industrial strain L-9.
 +
<br>
 +
<br>
 +
 +
<h3>RESULTS</h3>
 +
<h4>Gel electrophoretic analysis</h4>
 +
[[File:ZJU-CHINA_orfX_gel.png|500px|thumb|left|Fig.2 Gel electrophoretic analyses of PCR products (A) and selected examples of cloned products of seamless assembly reaction (B)]]
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
 +
<br>
 +
In Fig.2,  (A) 5-μl samples of the PCR products for orfX, (B) 5-μl samples of the bacteria solution PCR products were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. The DNA size standards was the DL1,000 DNA Marker (M1; Takara, Cat#3428A) and DL2,000 DNA Marker (M2; TaKaRa, Cat#3427A) . Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager. PCR products, positive clones and negative clones are indicated.
 +
 +
<h4>DNA sequencing</h4>
 +
We have sequenced the parts with standard primers VF2 and VR. The sequence of the 942bp part shows 100% agreement with the desired sequence.
 +
<br>
 +
<h3>REFERENCE</h3>
 +
Hwang, Y. S., et al. (2003). "Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various Streptomyces avermitilis Strains." Appl Environ Microbiol 69(2): 1263-1269.
 +
<br>
 +
<br>

Latest revision as of 16:09, 16 September 2015

orfX (from Streptomyces avermitilis, increasing avermectin production)


The part orfX is a putative membrane-bound putative regulatory gene and its product is a putative membrane protein.

This gene sequence could not function in E.coli. If you would like to express frr in Streptomyces avermitilis, remember to add ermEp [1] as its promoter.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 160
    Illegal NotI site found at 247
    Illegal NotI site found at 361
    Illegal NotI site found at 595
    Illegal NotI site found at 745
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 510
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 463
    Illegal AgeI site found at 264
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 441
    Illegal BsaI.rc site found at 195


Characterization

BACKGROUND

orfX is a putative membrane-bound putative regulatory gene and its product is a putative membrane protein.

Function

The results of PCR analysis and the gene disruption experiments strongly suggest that either a considerably conserved sequence of orfX or a combination of orfX and other assisting genes exists in the high producers. And the orfX product appears to play an essential role in the production and regulation of avermectin in both the normal strain and the high producers. When wild-type S. avermitilis was transformed with a 8.0-kb DNA fragment containing the orfX gene, avermectin production increased approximately 3.5-fold. However, the nature of the stimulatory effect of orfX is still unclear.

Principle

The orfX gene reveals a “copy number effect”. That is to say, multiple fragment copies can substantially increase avermectin production in S. avermitilis. Different from metK and frr gene, the DNA fragment containing orfX gene also increased avermectin bio-synthesis in various S. avermitilis strains, including the high-producing mutant strain ATCC 31780 and a semi-industrial strain L-9.

RESULTS

Gel electrophoretic analysis

Fig.2 Gel electrophoretic analyses of PCR products (A) and selected examples of cloned products of seamless assembly reaction (B)



















In Fig.2, (A) 5-μl samples of the PCR products for orfX, (B) 5-μl samples of the bacteria solution PCR products were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. The DNA size standards was the DL1,000 DNA Marker (M1; Takara, Cat#3428A) and DL2,000 DNA Marker (M2; TaKaRa, Cat#3427A) . Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager. PCR products, positive clones and negative clones are indicated.

DNA sequencing

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 942bp part shows 100% agreement with the desired sequence.

REFERENCE

Hwang, Y. S., et al. (2003). "Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various Streptomyces avermitilis Strains." Appl Environ Microbiol 69(2): 1263-1269.