Difference between revisions of "Part:BBa K2275010"

(→‎Information contributed by NFLS_Nanjing (2022))
 
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Biology and usage:
 
Biology and usage:
  
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1. The gene bpsA is expressed in C. glutamicum and carries strong fluxes toward L-glutamate, a precursor of indigoidine. [1]
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The gene glnA encodes the GS enzyme, which is very critical for synthesizing the L-glutamine inside the organisms. The GS enzyme not only provides the glutamine for the bacteria, but also functions as a nitrogen source, promoting the carbon and nitrogen metabolism.  
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+
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2. The gene bpsA catalyzes indigoidine formation from two molecules of glutamine in an ATP-dependent manner.[2]
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Experimental approach:
 
Experimental approach:
  
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1. Integrated sfp into the yeast chromosomal δ-sequences.
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1. Follow the instruction of the kit to extract the glnA gene from the fluid of C.glutamicum.
 +
 
 +
2. Amplify the gene glnA by PCR. The two primes are 5’-AAGCTTTCAATGAGGAGTCACCGTGGCGTTTG-3’ and 5’-GAGCTCCGGCTAGCTAAGTGAATTAGCAGTCG-3’.(The underlined part are the Restriction Enzyme cutting sites, HindⅢ and Sac I) Condition: 94℃ 5 min;94℃ 30 s,62.5℃ 30s,72℃ 3 min,30 cycles ;72℃ 10 min.
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3. Objective gene recovery
  
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2. Codon-optimize Te bpsA gene for expression in S. cerevisiae and genomically integrated into locus ARS1014a under control of the TDH3 promoter and ADH1 terminator using a previously reported, cloning free Cas9 toolkit.
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4. Construct pCES-GOG plasmid as the vector of glnAY405F, odhIT15A, gdh
  
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3. Use the conventional lithium acetate method to perform transformations.
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5. Synthesize strong promoter PH36 to clone and overexpress glnAY405F to promote the transformation of L-glutamate to L-glutamine. It will also prevent the negative effect of adenylation that restricts the GS enzyme.
  
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4. Use 200 ng pCut_1014a and 500 ng of linear Donor DNA with 500 bp homology to the integration locus ARS1014a.[3]
 
  
 
===References===
 
===References===
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[1] High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes
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* High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes
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[2] Genome-scale metabolic rewiring improves titersrates and yields of the non-native product indigoidine at scale
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[3] Production efciency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae
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Latest revision as of 14:45, 18 September 2022

PIacI-B0034-K2275004 (glnA-gudB)

We combined the gudB and glnA gene into pSB1C3 backbone. By SDS-PAGE method, we test the expression of both proteins as well. The gel image shows below, where "WC" indicates whole cell, "S" for supernatant, "P" for pellet, and "C" indicates the control of sample without plasmid.

SDS-PAGE result of GlnA and GudB.png

We can see that the glnA and gudB both expressed in E. coli at the same time. Next, we want to test if the Biobrick can function to convert ammonium to glutamine, so we add different ammonium concentration and use kit to detect the glutamine concentration.

Functional test of glnA-gudB.png

The figure above is the functional test result. When merely adding ammonium, the concentration of glutamine is very low. However, if both harboring the plasmid and adding ammonium, the concentration if glutamine is higher.The second and third bar can show the ability of converting ammonium to glutamine. 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 675
    Illegal BamHI site found at 1309
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 322
    Illegal NgoMIV site found at 1088
    Illegal NgoMIV site found at 2454
    Illegal AgeI site found at 578
    Illegal AgeI site found at 2891
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1481


Information contributed by NFLS_Nanjing (2022)

Part information is collated here to help future users of the BioBrick registry.

Metadata:

  • Group: NFLS_Nanjing (2022)
  • Author: Wenxin Mao
  • Summary: Added information collated from existing scientific studies

Biology and usage:

The gene glnA encodes the GS enzyme, which is very critical for synthesizing the L-glutamine inside the organisms. The GS enzyme not only provides the glutamine for the bacteria, but also functions as a nitrogen source, promoting the carbon and nitrogen metabolism.


Experimental approach:

1. Follow the instruction of the kit to extract the glnA gene from the fluid of C.glutamicum.

2. Amplify the gene glnA by PCR. The two primes are 5’-AAGCTTTCAATGAGGAGTCACCGTGGCGTTTG-3’ and 5’-GAGCTCCGGCTAGCTAAGTGAATTAGCAGTCG-3’.(The underlined part are the Restriction Enzyme cutting sites, HindⅢ and Sac I) Condition: 94℃ 5 min;94℃ 30 s,62.5℃ 30s,72℃ 3 min,30 cycles ;72℃ 10 min.

3. Objective gene recovery

4. Construct pCES-GOG plasmid as the vector of glnAY405F, odhIT15A, gdh

5. Synthesize strong promoter PH36 to clone and overexpress glnAY405F to promote the transformation of L-glutamate to L-glutamine. It will also prevent the negative effect of adenylation that restricts the GS enzyme.


References

  • High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes