NitroGENIUS: Engineering E. coli to Fix Nitrogen and Regulating Transcription with Light

By: Benjamin Huang1, Caroline Focht1, Hongyi Richard Li1, Jeffrey Lee1, Andrew Ng2, Bertram Berla3, Cheryl Immethun3, Deng Liu2, Tae Seok Moon3, and Himadri B. Pakrasi2,3, iGEM, Washington University in St. Louis1 Dept. of Biology, Washington University in St. Louis2 Dept. of Energy, Environmental and Chemical Engineering, Washington University in St. Louis3

                          

Sigma-Aldrich congratulates Team NitroGENIUS on their Silver Medal at iGEM in Boston, 2014. The International Genetically Engineered Machine competition (iGEM) is the premiere undergraduate Synthetic Biology competition where teams use a kit of standard parts to build unique biological systems.

Abstract

Endosymbiotic theory states that chloroplasts in plants originally came from a symbiotic relationship between prokaryotes. A certain cyanobacterium, Cyanothece sp. 51142, can photosynthesize and fix nitrogen within the same cell. Another photosynthetic cyanobacterium, Synechocystis sp. 6803, has light activated proteins. Our project is to study these nitrogen-fixing genes in a simpler environment such as E.coli and to work on a light repression system for gene expression. We want the nif cluster to be expressed when photosynthesis is not occurring, as oxygen poisons the nitrogenase enzyme.

Background

Cyanothece sp. 51142 can photosynthesize AND fix nitrogen within the same cell through a metabolic process where nitrogen fixation only occurs when photosynthesis is not active [1], minimizing oxygen interference.

Synechocystis sp. 6803 is a photosynthetic cyanobacteria that currently cannot fix nitrogen, but has been identified as a model cyanobacteria for future implementation in to the chloroplast. It has light sensitive proteins that regulate gene expression.

nif-cluster

Figure 1: nif cluster of Cyanothece sp. 51142 with all necessary genes for nitrogen fixation

strengthsofvarious-promoters

Figure 2: Modified from [2]. Ptrc1O is a strong hybrid promoter in both E.coli and Synechocystis

histidine-kinase-response

Figure 3: CcaR/CcaS (Green) light activated histidine-­kinase response system [3]

E.coli is a much simpler organism than cyanobacteria to study and manipulate: it is able to reproduce and grow rapidly, can survive in variable growth conditions, and is easier to genetically modify than cyanobacteria.

Wild type E. coli strains do not have native light regulation. Genes from Synechocystis sp. 6803 have previously been transferred to E. coli successfully, but light induction using green‐light activated CcaR/CcaS proteins show low dynamic range [4]. Swapping promoters has shown to be an effective means for increasing fold change, and hybrid promoters such as Ptrc1O are widely used.

Objectives

  • Determine the optimal conditions for culturing E.coli strains containing the Cyanothece sp. 51142 nif cluster
  • Select best strains for further testing
  • Create a light repressed gene regulatory mechanism
  • Compare fold change of light induction with new hybrid promoter

Results

nitrogenase

Figure 4: Nitrogenase Activity of 2 strains with and without the nif cluster

experimental-constructs

Figure 5: Experimental constructs and controls w/ expected fluorescence under light conditions

fluorescene-intensity

Figure 6: Fluorescence Intensity: Absorption: OD 600nm, Gain: 90, λex. 495nm/ λem. 528

Methods

  • Insert Cyanothece sp. 51142 nif cluster into various E. coli strains
  • Vary growth conditions including carbon source, nitrogen source, incubation temperature, and O2 concentration
  • Co-­transform plasmids w/ chromophore to activate light sensor
  • Fluorescence intensity measured with plate reader.

Conclusions

  • Of the five E.coli strains transformed with the nif cluster, JM109 and WM1788 showed strongest nitrogenase activity.
  • The linear relationship between nitrogen fixation activity and time matches that seen in nature.
  • Optimal conditions: glucose as carbon-­source, glutamate as nitrogen-source, LB as inoculating media, minimal M9 as testing media for GC assay, anaerobic environment at 30 °C for overnight preparation before acetylene reduction assay.*
  • PcpcG2 and hybrid promoters are leaky in the dark
  • Hybrid promoter completely turns off transcription in the light
  • Hybrid promoter seems to have greater dynamic range

*EnPresso® B Growth System was used for the culture of E. coli, achieving a 22% increase in cell density over LB.

Future Directions

  • Alter conditions to increase activity in JM109 and WM1788
  • Determine a minimal nif cluster
  • Clone positive controls of PcpcG2 and hybrid promoter with EYFP to verify that the hybrid promoter is light inducible
  • Weaken the RBS of TetR to reduce leakiness of promoters and eliminate dependency on aTc to see fold change
  • Swap out the reporter protein with the nif cluster
  • Transition into cyanobacteria by transferring the genes within the nif cluster into Synechocystis sp. 6803

References & Acknowledgements

  1. Welsh, et al. PNAS., 2008 September 30; vol. 105 No. 39
  2. Huang, et al. Nucleic Acids Research, 2010 Vol. 38 No. 8.
  3. Hirose, et al. PNAS., 2008 July 15; vol. 105 No. 28
  4. Tabor, Levskaya, Voigt. J. Mol. Biol., 2011 405: 315-324

 

We give thanks to our funding sources, the National Science Foundation MCB and following sponsors as well:

 

washu-2014-team

Team NitroGENIUS, from left to right: Richard Li, Jeffrey Lee, Caroline Focht, Benjamin Huang.

Materials

     
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