Part:BBa_K2326005

LacI - pTac - gadA + 6xHis Tag - Terminator

We also designed another way to testify whether gadA was expressed: polyhistidine-tag, an amino acid motif in proteins that consists of at least six histidine (His) residues. With polyhistidine-tag we can separate GadA out depending only on the structure of polyhistidien and justify whether the gene gadA was transcript in a large amount.

Introduction

Fig. 1 Third Approach: BBa_K2326005 & BBa_K2326006 We added a His tag after gadA so we can extract the enzyme out and prevent it from being subjected to endogenous processes, and then produce GABA from the isolated protein.

The First Test

1. Amino Acid Analyzer Result After Protein Purification

We purified GAD in E. coli Nissle and did the reaction. Fig. 2 – Fig. 4 show the result of reaction products. Since there were still no peaks in the range of 48 – 50 min, GABA was still not produced.

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Fig. 2 Yield of GABA of E. coli Nissle (RPG-BBa_K2326005) with reaction pH 4.2 (protein purification)



Fig. 3 Yield of GABA of E. coli Nissle (RPG-BBa_K2326005) with reaction pH 4.4 (protein purification)

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Fig. 4 Yield of GABA of E. coli Nissle (RPG- BBa_K2326005) with reaction pH 4.6 (protein purification)

When we analyzed the experiment, we found that we ignored the effect of cofactors. Pyridoxal phosphate is the cofactor of GAD enzyme, which is derived from vitamin B6 (Purves & Williams). In our reaction system, vitamin B6 was absent, so GAD might have low activity. We developed the next step based on this problem.

The Final Threshold

– Amino Acid Analyzer Result After Crude Enzyme Extraction

Since vitamin B6 presents originally in wild-type E. coli Nissle (Klijn), we decided to use crude enzyme extraction (Link to experiment page), so the cofactor of GAD was present in the reaction system. The same procedure was used to produce GABA. Then, the yield of GABA was measured by amino acid analyzer. Fig. 5 – Fig. 7 show the yield of GABA.

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Fig. 5 Yield of GABA of E. coli Nissle (RPG- BBa_K2326005) with reaction pH 4.2 (crude extraction)



Fig. 6 Yield of GABA of E. coli Nissle (RPG- BBa_K2326005) with reaction pH 4.4 (crude extraction)



Fig. 7 Yield of GABA of E. coli Nissle (RPG- BBa_K2326005) with reaction pH 4.6 (crude extraction)

The presence of peaks corresponding to 48 to 50 min suggests GABA was produced.

The yield of GABA is calculated by:
`"Peak area of GABA yielded by E.coli Nissle"/"Peak area of pure GABA sample"**"Concentration of pure GABA sample"**"dilution factor"`. Peak areas were derived from the system report of amino acid analyzer. Concentration of pure GABA sample in this experiment was 500 mg/L, and the dilution factor was 60. Fig. 17 shows yield of GABA at 3 pH values.



Fig. 17 Yield of GABA (after crude enzyme extraction)

The maximum GABA yield was 2976.79 mg/L, produced at pH 4.4. The modelling result suggests that the optimum reaction pH is 4.28, which is close to the optimum pH value in experiment. The result demonstrates that the optimum working temperature for GAD ranges from 4.2 to 4.6.

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 1720
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1443
  Illegal AgeI site found at 1522
  
• 1000
  COMPATIBLE WITH RFC[1000]