Part:BBa_K2418000

Lactate-dehydrogenase gene (LDH) from Bacillus coagulans

We needed an enzyme to catalyze the pyruvate-lactate conversion, therefore, we searched for the lactate-dehydrogenase (LDH). Since the subject of this study is the Methylococcus capsulatus (Bath) bacterium, which is a thermophilic organism (lives optimally at 45 oC), we thought, it would be worth searching for the sequence of an LDH gene of a thermotolerant organism as well. For this, we used the European Bioinformatics Institute’s database (www.ebi.ac.uk) in order to accomplish the search, and as a result, we found that the bacterium Bacillus coagulans 36D1 has an LDH enzyme, and lives optimally at 50 oC. The LDH sequence of the bacterium Bacillus coagulans 36D1 was found using the KEGG PATHWAY Database (http://www.genome.jp/kegg/pathway.html). Opening the enzyme’s datasheet, we realized that two enzymes are in connection with this reaction. We chose the L-lactate-dehydrogenase because it was smaller in size and had the usual beginning with ATG bases.

The LDH gene was intended to ligate between the NcoI and EcoRV restriction sites, therefore, it was supplied with an NcoI restriction site and an addition, non-coding AGTCAGTC nucleotide sequence before and after the NcoI restriction site, in order to minimalize the possible damage made by the restriction enzymes. The LDH gene contained an NcoI restriction site in itself, therefore, we replaced the bases so that the coded amino-acid did not change.

The figure of the lDH sequence

1. Addition AGTCAGTC bases

2. NcoI restriction site

3. LDH gene

4. EcoRV restriction site

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 110