Part:BBa_K2326004

Overexpression of gadA with super ribosomal bonding side controlling with lactose inducible promoter

Lactose-inducible lac operon transcriptional repressor (LacI) is a DNA-binding transcription factor that represses transcription of the operon involved in transport and catabolism of lactose. ("lacI lactose-inducible lac operon transcriptional repressor [Escherichia coli str. K-12 substr. MG1655] - Gene - NCBI", 2017) The pTac promoter is a functional hybrid promoter, derived from the Trp and Lac promoters, that are regulated by Trp and Lac. Global non-modularity towards promoters & protein-coding parts and relative strength was estimated for RBSs B0030, B0032, B0034 in the research of Antiquity (2003-01-31). ("Part:BBa K180000 - parts.igem.org", 2009) Part BBa_E0040 is the green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212) designed by jcbraff in Antiquity (2004-09-30). ("Part:BBa E0040- parts.igem.org", 2017)

Introduction

http://2017.igem.org/File:Fig_2_Second_Approach_BBa_K2326004.png

Fig. 1 Second Approach: BBa_K2326004

To address the problems found in BBa_K2326003, we designed BBa_K2326004. Because E. coli Nissle has not been used as an engineered strain until recent years, it cannot express large amount of foreign genes from a plasmid and failed to execute all the designs we had on our first attempt. So we cut our original design, leaving the most important enzyme, gadA, alone, which catalyzed GABA transforming from glutamate. We also changed the RBS of gadA into B0034, the most consensus sequence for E. coli. Moreover, we added GFP at the end of the circuit to simplify the process of assaying protein production.

Fig. 2 Sequencing result of construction of RPG- BBa_K2326004. Derived from Snapegene.

Test

1. Plate Reader

The part contains green fluoresce protein (GFP). We measured the relative concentration of GFP in bacterial suspension to determine the expression of gadA. The concentrations of GFP in M9 broth are represented by `"Relative Fluorescence Intensity"=log⁡("Fluorescence Intensity")/("OD"_600)`in the figure below. Positive control was tested on E. coli Nissle with PSB1C3-GFP plasmid, and the negative control was tested on the endogenous, wild-type E. coli Nissle. The same induction process was used.

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Results suggest the concentrations of GFP expressed by E. coli Nissle with the constructed plasmid was higher than the positive control and negative control. Since gadA was closer to the promoter compared to GFP, it was expected to express more than GFP.

2. Amino Acid Analyzer



Fig. 3 yield of GABA of E. coli Nissle with part Bba_K2326004 (M9 broth)

The same experimental condition was used in the amino acid analyzing process. Fig. 3 shows the yield of GABA by E. coli Nissle with the constructed plasmid in M9. However, there were still no peaks during 48 min to 49 min in the two figures, indicating that GABA was not produced. </ol>

Problems

There were two possible reasons of our failure to produce GABA. First, there might be a strong ribosomal binding site located upstream of GFP, making most of the ribosomes bind with GFP but not gadA. Thus, gadA was not fully expressed. Second, evidence suggests GABA permease, 4-aminobutyrate aminotransferase, and 4-aminobutyrate aminotransferase presented in E. coli can degrade GABA in a catabolic pathway, so GABA maybe have been degraded prior to analysis (Vo & Hong).

Our culture conditions during induction had deficiencies as well. According to Lü et.al., the yield of GABA of L. sakei B2-16 after incubating for 4 days in MRS medium containing 3% MSG was 165 mM. Since we only incubated the bacterial culture for 12 hours and the concentration of MSG was 1%, E. coli Nissle might not have enough time and enough substrate to produce detectable GABA. Moreover, the GAD activity of Lactobacillus brevis NCL912 is the highest when pH of MSG medium ranges from 4.0 – 5.0 (Huang et.al.). However, the pH of LB medium is about 7, so GAD might not function well in such a basic environment. Therefore, the yield of GABA may have been low.

We developed Bba_K2326005 and following experiment to eliminate these problems.

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 1711
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1434
  Illegal AgeI site found at 1513
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3463