Results

The results of our work regarding sequencing, PRC and gel electrophoresis, confocal microscopy, and nitrogenase protien expression are detailed on this page. Our overall goal was to introduce the nitrogenase gene cluster into our E. coli cells by assemnbling the cluster in vivo using IVA assembly. We assembled the cluster first in three subsections: BHD cluster (nifB, nifH, and nifD), the KEN cluster (nifK, nifE, and nifN) and the XAV cluster (nifX, hesA, and nifV). Afterwards, we assembled these three subsections into our final gene cluster, containing the nine core genes necessary for nitrogen fixation in E. coli [1]. Concurrently, we introduced an activator and repressor plasmid into our cells as detailed in (Stricker et. al., 2008) [2]. We did so with the goal of using this positive and negative feedback system to acheive oscillating expression of both nitrogenase and GFP. In the future, we aim to repeat and refine our experiments, and with success, we aim to use our system to oscillate the expression of nitrogenase in other bacteria that may be better suited for agricultural use. It is our hope that our data will be useful for future iGEM teams interested in Sticker's feedback circuit, or in working with the nitrogenase enzyme.

Sequencing

For each of the plasmids that we sequenced, we cataloged the mutations we observed. In addition, we assessed whether or not the ligation was sucessful, or if we would have to attempt it again. When applicable, we documented when the plasmid that we sequenced was used in subsequent experiments.

See the example below:

Figure 1: Insertion of nif B, nif H, and nif D genes into the 2Bc-T plasmid vector using IVA (attempt #1)

Mutation

• Within the nif gene cluster:
  • 1 missense mutation in nif K (C to A mutation)
  • 1 silent mutation in nif E (G to A mutation)
  • 3 silent mutations in nif N (2 G to A, one C to G mutation)
• Within the nif gene cluster:
  • 1 missense mutation in nif K (C to A mutation)
  • 1 silent mutation in nif E (G to A mutation)
  • 3 silent mutations in nif N (2 G to A, one C to G mutation)

This ligation succeeded; all 3 nif genes were inserted in the correct order. This colony was used for subsequent assembly attempts.

PCR & Gel Electrophoresis

Firstly, we wanted to make sure that our PCR primers were functional in attaching the homology overhangs needed for either SLIC or IVA.

We found that all of our samples were able to be amplified with all of our IVA primers. For sake of convenience and maximizing the most help for future iGEM teams, we characterized the IVA primers into the parts pages (see our parts pages), and left the SLIC primers documented in a separate document.

However, despite all the primers designed with the same guidelines, we found that PCR troubleshooting was needed for each of the primer pairs. We primarily changed annealing temperature, annealing time, and number of cycles.

See the detailed work below:

Confocal Microscopy

To ensure replication of the genetic oscillator system, we decided to show these results through confocal microscopy. Although much troubleshooting is needed to fully show the clear oscillations, we did obtain preliminary results that were very promising. In the coming weeks, before the Grand Jamboree, we will work on troubleshooting this exact result to show distinct changes in GFP signals.

For preliminary results, and as a proof of concept that the oscillator is replicable, we show the following video and images to show that the GFP signal is dimming in the time period it should.

We tried to image for longer, however, long periods of exposure of the fluorescence signal to the E. coli cells showed that the GFP signal attenuated very rapidly (within 30 minutes or so). We will be working on showing results that will be independent of this confounding variable, to show that the GFP signal both dims and starts to brighten up again, with reliable periods.

Figure 2: Confocal microscopy results, time period: 3:32:58

Figure 3: Confocal microscopy results, time period: 3:39:28

Despite severe attenuation of signal of GFP, this cell, which remained in solution near the same spot all throughout (verified by brightfield image, although not imaged here), showed an increase in GFP signal.

We will improve upon these results after the wiki freeze, to show clearer oscillations and link full results with dry lab models and predictions.

Nitrogenase Protein Expression

Finally, we ran a Coomassie stain with overexpressed protein concentrations. We expressed each of the three constructs: BHD, KEN, and XAV. This particular sample read from right to left: protein ladder, pre-induction BHD, 3 hours BHD, 9 hours BHD, pre-induction KEN, 3 hours KEN, 9 hours KEN, pre induction XAV, 3 hours XAV, and 9 hours XAV. We also found bands at possibly the right size, and protein concentrations increased with time. Unfortunately, we realized we had to troubleshoot to make sure that these bands are resolved much better. We plan to troubleshoot these results between the wiki freeze and the final Jamboree presentation.

Proof of Concept

Based on our compiled results; we realize the following proofs of concepts:

1. We showed that oscillations occur in our system; with confocal microscopy and dry lab modeling.


1. We had hoped for a very strong connection between dry lab’s model and wet lab’s replication and imaging of the oscillator. Regardless, we were able to show evidence of an oscillation given the level of arabinose and IPTG used.


2. We showed that there was some evidence of complete assembly of the minimal nif operon.


1. First, we saw a 10kb product when we amplified the 2Bc-T vector with T7 forward and reverse primers.


1. Although our sequencing results showed a majority product for another plasmid, with a smaller size (which we later characterized as 2Bc-T with XAV inserted inside), we found a very small number of plasmids that were around the size we wanted to see.
   
   



2. We tried amplifying this construct with T7/T7 terminator primers. We saw the following gel results. We will attempt to gel extract this band, and use it for PCR to see if the whole operon can be obtained from this construct.

   
3. We obtained colonies (albeit very low CFU) with JM109 DE3 transformed with the miniprep colony of the above gel result on nitrogen-free media.


1. Although further screening must be done: there were two colonies on a 100 mm plate with cells induced with 1mM final concentration IPTG. We hope this means our expression of nitrogenase was successfully complete, but further tests will be done after the wiki freeze.