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Using an inducible hybrid light-sensitive promoter for heterologous regulation of the nif cluster

Benjamin Huang, Jeffrey Lee

Introduction

Synthetic biology is an exciting area of research that aims to genetically improve organisms to make them more efficient and hopefully more useful to us as well. The human population in 1950 was 2.5 billion, yet it is predicted to surpass 9 billion by 2050 [1]. Even with population growth slowing, increasing life spans and standards of living will soon tax our natural resources. One of the most concerning is our food supply. The agriculture industry needs a revolution in order to keep up with our expected growth rates. Currently about 80% of chemically fixated nitrogen is used as agricultural fertilizers, the majority in developed lands [1]. Intracellular nitrogen fixation in crops could help to sustain the burgeoning world population, especially in areas with less fertile soil without taxing the planet’s waterways. The exponential increase in nitrogen fertilizer has led to more runoff into rivers and oceans. Fertilizers then provide nutrition for algal blooms that result in hypoxia and form oceanic dead zones. These dead zones lead to the death of marine species and have potentially large economic consequences.
The ramifications of nitrogen fertilizer runoff can be averted by genetically engineering plant crops to fix their own nitrogen. Some cyanobacteria fix nitrogen for nutritional needs, while most organisms can only acquire it from the food it consumes. Synthetic biology allows us to transfer this ability to fix nitrogen to a heterologous host that has many genetic tools, Escherichia coli, so that we can learn how to give single cell organisms, and eventually chloroplasts the ability to create their own nitrogen fertilizer.
Diazotrophic (organisms that fix nitrogen) cyanobacteria such Nostoc Punctiforme or Anabaena use heterocysts (specialized nitrogen fixing cells) to create a mini-anaerobic environment to aid nitrogen fixation. However, Cyanothece 51142, a non-heterocyst, fixes nitrogen in the same cell as photosynthesis by relying on a circadian metabolic process, when there is less oxygen byproduct from photosynthesis. Our goal this summer is to engineer the regulation of the proteins necessary for nitrogen fixation so that they are highly repressed when activated by broad spectrum light (such as the sun), and are highly active when there is no light around, mimicking the cycle where photosynthesis occurs during the day and nitrogen fixation occurs at night.
Traditionally, gene expression has been induced through chemical means. However, this method requires expensive chemicals, and can have deleterious side effects on cell health. In Tabor et. al (2010), it was shown that it is possible to induce expression of a reporter gene with light in E. coli using light sensitive proteins (CcaR/CcaS) from the model cyanobacterium Synechocystis S. 6803. Light induction has several advantages over chemical induction, including: long term cost of chemical inducers, ability to tune expression levels with different light intensities, and most importantly, can be turned on and off easily. The ability to control genes of interest has important applications in engineering nitrogen fixation. The end goal of our project is for plants to fix nitrogen on their own within their chloroplasts--the true powerhouse of plant cells, where the most ATP is present to overcome the high costs associated with breaking nitrogen’s triple bond.

Objectives

Synechtocystis PCC6803 (hereafter S. 6803) is a strain of cyanobacteria that is of particular interest to us. S. 6803 is important because it is the first cyanobacteria to have its complete genome sequenced; and it is easily manipulated via homologous recombination. Another aspect of S. 6803 that renders it a relevant is based on the ultimate goal of allowing plants to fix their own nitrogen. Nitrogen fixation in these genetically engineered plants would have their own cellular machinery (analogous to chloroplasts and mitochondria) that direct the evolution of nitrogen. Based on the Endosymbiotic theory, chloroplasts and mitochondria are thought to have been cyanobacteria that were engulfed by cells. Thus, S. 6803 is an important platform from which further manipulation of the nitrogen fixation can occur.
Recently it has been shown that one can induce via green light, the expression of a phycobilisome-related gene [2]. Instead of regulating gene expression via proteins, having to worry about different concentration levels, potential cross effects, etc., regulation via light can be easily tested for and manipulated as well. Therefore, we propose to design a hybrid inducible light-sensitive promoter for heterologous regulation of the nif cluster in E. Coli.

Approach

We created a 4 piece assembly plasmid integrating light regulation components from pJT122, but swapping out cph8 (for EYFP from pSL2264) and lacZ (for TetR from pTet-PP*) and combined them into a plasmid PBJ003 which should repress expression of EYFP when induced by light. PBJ003 is controlled by the basic cpcG2 promoter driving TetR production. We also created a hybrid promoter to swap out for the basic cpcG2 promoter. Both these parts are on the registry under BBa_K1385000 and BBa_K1385001.

Hybrid promoters that integrate the regulatory elements from several sources have been shown to be functional with varying levels of success [3]. Operator sites for the green-light sensitive PcpcG2 promoter are well known [4], additionally, the hybrid trc1O promoter has been shown to be highly expressed in E. coli (22-fold induction) even without IPTG [5]. The trc1O promoter has one lac operator site and is thus easier to induce than the more tightly repressed trc2O that contains two repressor binding operator sites.

In order for PcpcG2 to function as a light-sensitive promoter, a histidine kinase CcaS and response regulator CcaR will be required. Upon exposure to green light, CcaS is auto-phosphorylated and then phosphorylates CcaR. This changes the DNA binding affinity to CcaR allowing CcaR to bind to PcpcG2 and activate transcription. This could be obtained by creating our own plasmids with these genes (already done). Our hybrid promoter could be cloned into a vector that contains a fluorescent reporter protein EYFP driven by EYFP to compare the strength of transcription when activated by light in E. coli containing both CcaS/R, eventually allowing the light promoter to regulate the nif cluster.

A few factors have been taken into consideration for the design of the hybrid promoters. The consensus sequence known as the “G-box”, a highly conserved direct-repeat motif located 109 and 108 bases upstream of the transcription start site in N. Punctiforme and S. 6803, is a region where CcaR binds to PcpcG2 as an activator [4]. We choose to use Ptrc1O and keep the whole sequence intact because it is shown to be highly repressible and easily inducible and thus should essentially work as a constitutive promoter in E. coli, even though it has been reported to be leaky in S. 6803 [5]. By combining the upstream components (CcaR binding site) of PcpcG2 with Ptrc1O, while keeping the Ptrc1O ribosomal binding sequence intact, our hybrid PcpcG2/trc1O promoter in E. Coli should be highly expressed in (green) light with basal levels of transcription without lig

As described above, the hybrid promoter functions the opposite of how we eventually want it to; namely, we want nitrogenase to be highly repressed in light conditions (where a lot of oxygen will be present to poison nitrogenase), and highly expressed in the dark. Therefore we need to create a genetic switch that functions essentially as a NOT gate, where in high light conditions, our product (currently EYFP), will be highly repressed.
Different switches based on RNA have been well documented in E. coli; we have several options. First we are testing the repression mechanism using a basic pTet/TetR, repression mechanism. By expressing TetR, the system driven by the hybrid promoter should show strong repression in light, and strong expression in dark.

Our next step would have been to swap out the pTet/TetR repression with a system of CRISPR interference to block transcription initiation or elongation based on binding to the template or non-template strand of a chosen sequence [9]. In our case, we would tune the sgRNA to bind to the -35 box of our hybrid promoter in order to block transcription. The reason we want to use CRISPRi is because it has been shown to be functional in both prokaryotes and eukaryotes, although slightly more complex, this becomes a much more powerful tool once people transition to eukaryotic environments. Both of these genetic switches should be able to highly repress our fluorescent protein when induced with light.

Meanwhile, once the other half of our iGem team gets nif cluster working in E. coli, we should be able to swap out the fluorescent protein for the nif cluster in our plasmid to regulate the nif cluster in E. Coli with light. In dark conditions, the switch will be “off”, namely there should be fluorescence; and in the light, the system should be repressed. We expect to see higher levels of repression with our hybrid promoter and greater dynamic fold change.

Our project goals are to design a hybrid promoter that incorporate the regulatory elements of PcpcG2 with those of Ptrc1O. Then we will transition to the more complex CRISPRi mechanism by creating a plasmid containing the basic trc1O promoter that reversibly blocks transcription of EYFP. The overarching plan is to be able to regulate the nif cluster with light, so nitrogenase is only expressed during the dark, which mimics Cyanothece 51142. If successful, this project would help immensely when eventually integrating nitrogen fixation into plants by improving efficiency. During the day, when photosynthesis occurs (with a lot of oxygen as byproduct) nitrogenase would not be produced, and therefore not wasted; while during the night, nitrogen fixation can occur. This creates a temporal regulatory system orthogonal to Cyanothece 51142’s native circadian regulation of controlling nitrogen fixation (through metabolic pathways), increasing the overall chance of success.

Condition	Product	Fluorescence
Light	TetR	Basal
Hybrid Light	TetR	None
Dark	EYFP	High

By integrating the light regulation from S. 6803 into E. coli, we are transitioning to the next step in our big picture, which would be to get the system working in the cyanobacteria S. 6803. Eventually we want our system to be able to work in chloroplasts of plants, so nitrogen fixation will be able to occur in the same location where the most energy is produced without having to worry so much about the oxygen byproduct of photosynthesis.

Summer PROPOSED Work Flow

Week 1: Familiarize self with standard laboratory methods and procedures. Design primers for transcriptional units and plasmid backbone.
Week 2-3: Test light regulation in pBJ003 (driven by basic PcpcG2). Use positive and negative controls as well to ensure system is working as intended with EYFP fluorescence as our gene expression.
Week 4-5: Swap out basic PcpcG2 in pBJ003 with our hybrid promoter and test for increased Fluorescence of EYFP.
Week 6-8: Swap out tetR/ptet with CRISPRi mechanism with dCas9 and sgRNA plasmids. If things go as planned, swap out EYFP with nif cluster in hybrid promoter driven plasmid.
Week 9-10: Analyze data, comparing fluorescence in different intensities of light, wavelengths, time intervals, etc.

Methods

Our general workflow for each plasmid cloning is as follows:
a. Primer design for backbone and pieces
b. PCR pieces for amplification
c. Run a gel extraction
d. Gel DNA Recovery
Treat with DpnI and purify if necessary
e. Digestion/Ligation (Golden Gate or Blunt End)
f. Transformation (Electroporation)
g. Plate and grow overnight
h. Pick colonies and start liquid culture overnight
i. Freeze and Miniprep cultures
j. Run sequencing PCR to check junctions
k. Run visualization gel for sequencing PCR
l. Send for sequencing
m. Verify sequence products are as intended
With every step there is a chance that things could go wrong, and we would troubleshoot in order to determine the best course of action going forward.

Data

Figure above: Original Light induction experiment once all the cloning was over. No fluorescence seen.

After all our cloning mishaps we finally were able to test our light induction system. However, nothing worked as expected. The data represented above is the fluorescence normalized to the absorbance, or OD of the strains we grew. We essentially had the same fluorescence as our negative control, and even when we induced with 250ng/mL of ATC we saw no fluorescence. This led us to run a few more experiments to troubleshoot the plasmids.

Figure above: We ran another crude experiment to check what could be the issue.

Our original positive control was under control of constitutive promoter, J23100, and was the only thing we got to fluoresce. We were concerned with competing translation start sites so we cloned a plasmid that modified the RBS to allow for EYFP to fold properly, but fluorescence only got weaker. We then decided to swap out the pTet promoter and lo- and behold got fluorescence. Our next step was to swap out the reporter mechanism of our experimental plasmids.

Figure above: Fluorescence in the dark (when promoter not active) same regardless of chromophore presence.

Figure above: Fluorescence in the light (when repressor active) shows some difference with aTc present. Hybrid leaky.

Results

Figure above: Final Light experimental data

Normalized fluorescence is compared to the positive control after subtracting the negative fluorescence value. The Hybrid 0 aTc results in light ended up fluorescing even less than the negative control, implying the system is completely off, which is what we ideally want.

Another thing to note is that the system isnt working 100% as expected, but it is still promising.
When comparing light to dark values, the general trend is there, but the repressor is very leaky. There is so much tetR in around that it is dominating the system; with no aTc around, there is so little fluorescence, but once we add a meaningful amount of aTc, we are able to see results closer to what we expected. In the dark, we want our fluorescence to be relatively high, comparritive to the positive control, and we want the system to be highly repressed in the light (without having to induce with aTc).

When we compare the hybrid promoter to the normal cpcG2 promoter, it seems like there is a bigger change between dark and light in the system. In constant light, aTc is known to degrade, which is why some of the fluorescence might still be there. Hence, in the light we do not know how much of the repression is due to the strength of the promoter, or the reduction of aTc. It is apparent that the hybrid promoter is leakier than the original- even with a high concentration of aTc we see very little fluorescence in the light. A comparable amount of aTc would have degraded in the light of both the hybrid and normal promoter, but it is hard to say if the difference in both light values with aTc would have been as apparent without the degradation of aTc.

Our next steps would be to modify the RBS of TetR in order to find a saturation point where the system is most tightly on and off without aTc. We ideally do not want to use aTc at all, as chemical induction is troublesome and won't be very reliable if degraded by constant light.

After we modified the system to be working without aTc around, we can look into swapping EYFP with the nif cluster to have the genes expressed only in the dark.

Citations

1. Rogers, Oldroyd. Journal of Experimental Botany March 2014 doi:10.1093/jxb/eru098
2. Hirose, Shimada, et al. PNAS July 15, 2008 vol. 105 no.28
3. Lutz and Bujard Nucleic Acids Research, 1997, Vol. 25, No. 6
4. Hirose, Narikawa, et al. PNAS May 11, 2010 vol. 107 no. 19
5. Huang et al. Nucleic Acids Research, 2010 Vol. 38 No. 8
6. Tabor, Levskaya, et al. J. Mol. Biol. (2011) 405, 315-324
7. Kim, White, et al. Molecular Systems Biology 2006 doi:10.1038/msb4100099
8. Brantl, Wagner. J. Bacteriol. 2002, doi:10.1128/JB.184.10.2740-2747.2002.
9. Larson , Gilbert, et al. Nature, Oct. 2013 vol. 8 no. 11
10. Abe, Koichi, et al. Microbial biotechnology (2014).

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 Normalized fluorescence is compared to the positive control after
 subtracting the negative fluorescence value. The Hybrid 0 aTc results

Team:WashU StLouis/Project/light