Part:BBa_K2326003

LacI - pTac - gadR - gadC - gadA

In the natural state, the arrangement of gadA and gadC is close to each other in the genome, ensuring the timely co-regulation of transcription and translation of gadA and gadC for GABA production. GadCA forms an operon and is regulated by gadR, which had much higher expression levels. (Li, Li, Liu & Cao, 2013)

Introduction

Fig. 1 First Approach: BBa_K2326003

We chose the pTac promoter, a hybrid of Lac and Trp promoters, induce GABA production in E. coli Nissle in the human intestine in the presence of lactose from consumed milk. The promoter can be induced by both lactose and IPTG, where LacI represses transcription of up stream genes in this part, which are gadR, gadC, and gadA. The ribosomal binding site (RBS) for gadRCA with a relative strength of 300, 300, 3900 respectively are calculated by RBS Calculator from Salis Lab.

Fig. 2 Sequencing result of construction of RPG- BBa_K2326003. Derived from Snapegene.

Test

1. SDS-PAGE

We test the expression of glutamate gamma-aminobutyrate antiporter and glutamate decarboxylase (GAD), which were coded by gadC and gadA respectively. Fig. 2 shows the result of SDS-PAGE. The protein size of the antiporter should be 55.22 kDa, and for the GAD enzyme should be 52.91 kDa. Protein bands in the correct molecular weight range were visualized in the area around 50 kDa. Comparing the control group, which used endogenous, wild-type E. coli Nissle, the protein bands presented in the supernatant of E. coli Nissle liquid culture with constructed plasmid were clearer, indicating that gadC and gadA are expressed.



2. Amino Acid Analyzer

We also tried to measure the yield of γ－aminobutyric acid (GABA) by using amino acid analyzer (AAA). Firstly, analytically pure GABA sample solution was tested and the result is shown in Fig. 3. The peak of GABA should have reaction time of 48 min to 49 min, according to the standard control run.

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Fig. 3. Amino acid analyzer result of pure GABA sample

Since the promoter we used is pTac, inducible by Isopropyl β-D-Thiogalactoside (IPTG), the IPTG inducing concentration was 1.0 mM. 1% monosodium glutamate (MSG) was added as substrate. After 12 hours’ induction, the bacterial supernatant was processed and tested. Fig. 4 shows the result of the GABA yield by E. coli Nissle with the constructed plasmid. However, there was no peak during 48 min to 49 min, indicating that GABA was not produced.

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Fig. 4 yield of GABA of E. coli Nissle with part Bba_K2326003

Problems

There were three possible problems in our construction causing the inability to produce GABA. First, since we were unable to know which genes the transcriptional regulator (gadR) regulates in Lactobacillus brevis NCL912, gadR might not function well in E. coli Nissle because of the absence of genes it regulates. Also, we could not locate the glutamate gamma-aminobutyrate antiporter (gadC) in E. coli Nissle cell membrane, so gadC might not function. There were two genes between the pTac promoter and gadA, so gadA might not be fully expressed due to increased plasmid and gene lenth. Based on the analysis of these problems, we constructed the part Bba_K2326004.

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 3863
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3586
  Illegal AgeI site found at 3665
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4973
  Illegal SapI site found at 2505