Difference between revisions of "Part:BBa K2936004"

 
 
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<partinfo>BBa_K2936004 short</partinfo>
 
<partinfo>BBa_K2936004 short</partinfo>
  
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lbfdh is a gene sequence expressing formic dehydrogenase. The formic acid dehydrogenase expressed by lbfdh oxidizes formic acid to carbon dioxide. This sequence can be used after flhA.
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This part is the coding sequence for formate dehydrogenase. Formate dehydrogenase can oxidize formate to carbon dioxide with the help of NAD coenzyme.
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===Usage and Biology===
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Since the accumulation of formic acid would affect the growth of the strain, in order to further optimize the degradation efficiency of FDS-2 for formaldehyde, we found that LBFDH existing in L. buchneri could degrade formate into CO2. By introducing the gene controlling LBFDH expression, hoping to reduce the accumulation of formic acid in bacteria and improve the degradation efficiency of formate. ( Degradation principle is shown in Figure 1, and the plasmid map is shown in Figure2)
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<center><img src="https://static.igem.org/mediawiki/parts/1/16/T--ZJUT-China--igem-zp1.png" width="700px"></center>
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<center><img src="https://static.igem.org/mediawiki/parts/3/3f/T--ZJUT-China--igem-zp2.png" width="500px"></center>
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===Characterize===
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In order to find an appropriate enzyme activity test method, after searching for literature and patents, we found a high-throughput screening method: first, we used the developer for preliminary screening, the formulation and concentration of the developer are shown in Figure 3，and the color rendering effect is shown in Figure 4.  
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<center><img src="https://static.igem.org/mediawiki/parts/6/6b/T--ZJUT-China--igem-zp3.png" width="400px"></center>
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Then we used different concentrations of NADPH to measure the OD<sub>340</sub> value, and a linear relationship was determined by plotting, so as to determine that the value of OD<sub>340</sub> was independent of ammonium formate and other concentrations. (the result is shown in Figure 6)
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Next, we added the substrate ammonium formate, NADP+, and crude enzyme solution into the VP tube, measured the OD<sub>340</sub>, and determined the appropriate concentration of the reaction system. (The result is shown in Figure 7)
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The concentration of different substrates (ammonium formate, NADP+) was used to measure the OD<sub>340</sub> of Lbfdh, and the Km and Kcat values were calculated respectively. (The result is shown in Figure 8)
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Finally, we did an enzyme assay. We calculated the values of Kcat and Km, and tested the enzyme activity of lbfdh gene, and set BL21 strain as the control group. Meanwhile, according to the defined enzyme activity formula, the enzyme activity of the experimental group and the control group was calculated and compared. Enzyme activity calculation formula = (OD340*50" dilution factor ")/(6.22" absorption coefficient per μmol NADH at 340 nm "*OD<sub>600</sub>" concentration of bacteria solution before crushing "*time" reaction time). The result is shown in Figure 9.
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===Experimental results===
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<html><center><img src="https://static.igem.org/mediawiki/parts/f/f9/T--ZJUT-China--igem-zp5.png" width="400px"></center></html>
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<br>
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In order to get the lbfdh fragment, we conducted plasmid PCR experiments, and the nucleic acid map is shown in Figure 5.
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<br>
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<html>
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<center><img src="https://static.igem.org/mediawiki/parts/1/18/T--ZJUT-China--igem-zp6.png" width="700px"></center>
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</html>
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There was a linear relationship between OD<sub>340</sub> and NADPH, and the influence of ammonium formate and other substances on OD<sub>340</sub> was basically ignored.
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<br><br>
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<html>
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<center><img src="https://static.igem.org/mediawiki/parts/7/78/T--ZJUT-China--igem-zp7.png"  width="700px"></center>
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</html>
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The appropriate results for each component are shown Figure 7. And the definition of IU：The amount of enzyme required to produce 1μmol NADPH in 1min.
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<br>
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<html>
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<center><img src="https://static.igem.org/mediawiki/parts/b/bb/T--ZJUT-China--igem-zp8.png" width="700px"></center>
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<br>
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<center><img src="https://static.igem.org/mediawiki/parts/1/18/T--ZJUT-China--igem-zp9.png" width=500px></center>
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</html>
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The above data proved that the LBFDH gene we imported had enzyme activity in host bacteria.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 01:55, 22 October 2019


Formate dehydrogenase(Regulated by lbfdh gene)

This part is the coding sequence for formate dehydrogenase. Formate dehydrogenase can oxidize formate to carbon dioxide with the help of NAD coenzyme.


Usage and Biology

Since the accumulation of formic acid would affect the growth of the strain, in order to further optimize the degradation efficiency of FDS-2 for formaldehyde, we found that LBFDH existing in L. buchneri could degrade formate into CO2. By introducing the gene controlling LBFDH expression, hoping to reduce the accumulation of formic acid in bacteria and improve the degradation efficiency of formate. ( Degradation principle is shown in Figure 1, and the plasmid map is shown in Figure2)

Characterize

In order to find an appropriate enzyme activity test method, after searching for literature and patents, we found a high-throughput screening method: first, we used the developer for preliminary screening, the formulation and concentration of the developer are shown in Figure 3，and the color rendering effect is shown in Figure 4.

Then we used different concentrations of NADPH to measure the OD340 value, and a linear relationship was determined by plotting, so as to determine that the value of OD340 was independent of ammonium formate and other concentrations. (the result is shown in Figure 6)
Next, we added the substrate ammonium formate, NADP+, and crude enzyme solution into the VP tube, measured the OD340, and determined the appropriate concentration of the reaction system. (The result is shown in Figure 7)
The concentration of different substrates (ammonium formate, NADP+) was used to measure the OD340 of Lbfdh, and the Km and Kcat values were calculated respectively. (The result is shown in Figure 8)
Finally, we did an enzyme assay. We calculated the values of Kcat and Km, and tested the enzyme activity of lbfdh gene, and set BL21 strain as the control group. Meanwhile, according to the defined enzyme activity formula, the enzyme activity of the experimental group and the control group was calculated and compared. Enzyme activity calculation formula = (OD340*50" dilution factor ")/(6.22" absorption coefficient per μmol NADH at 340 nm "*OD600" concentration of bacteria solution before crushing "*time" reaction time). The result is shown in Figure 9.

Experimental results


In order to get the lbfdh fragment, we conducted plasmid PCR experiments, and the nucleic acid map is shown in Figure 5.

There was a linear relationship between OD340 and NADPH, and the influence of ammonium formate and other substances on OD340 was basically ignored.


The appropriate results for each component are shown Figure 7. And the definition of IU：The amount of enzyme required to produce 1μmol NADPH in 1min.


The above data proved that the LBFDH gene we imported had enzyme activity in host bacteria.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 318
    Illegal BamHI site found at 30
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 300
    Illegal NgoMIV site found at 942
    Illegal AgeI site found at 1129
  • 1000
    COMPATIBLE WITH RFC[1000]