Team:Cadets2Vets/Results

The Arsenic 1.1 plasmids that Cadets2Vets worked with in June and July were synthesized by GeneWiz in a PUC57 vector. Preliminary experiments were performed using these plasmids until unexpected results in July forced us to examine the DNA more closely.

We analyzed the sequences that were sent to GeneWiz for synthesis and compared them against the NCBI BLAST database and the iGEM Registry, and found that the sequences sent to GeneWiz were not the sequences that we had intended to synthesize. The regulator we used appeared to be a generic transcription factor and the GFP had amino acid substitutions that were unexpected. We do not think the mutations affected GFP expression but because it did not match the Part in the Registry, we needed to correct that gene.

Cadets2Vets redesigned the plasmids using the appropriate Parts and successfully cloned synthetic DNA into the pSB1C3 vector backbone using the BioBrick Assembly method. The ligation of the inserts into the backbone was verified using restriction enzyme digest (left).

We screened 2 clones for every transformation performed during the cloning of the Arsenic Circuit. The DNA gel on the left shows the products from inserting PcArsR or PArsRGFP into the pSB1C3 vector. The plasmids were digested with the specified restriction enzymes and we expected PcArsR to create a 569bp fragment and PArsRGFP to create a 969bp fragment. The backbone is 2070bp.

The DNA gel on the right is from 2 clones where we attempted to ligate PcArsR and PArsRGFP into the pSB1C3 backbone. We used restriction enzymes to cut PArsR out of the vector, and found that the vector fragment is different sizes for the two clones we screened. We expected to have ~3000bp vector (2070 + 969bp), which is consistent with clone #1. Clone #2 is only ~2000bp, indicating that the PArsRGFP insert did not integrate. Clone #1 is the correct plasmid to keep.

Cadets2Vets tried a variety of different experiments to study the function of our circuit. We felt that we learned a lot of new scientific methods and saw that were were many different ways of approaching a problem scientifically. Our initial results suggested that the arsenic circuit was working, but we needed stronger data that was reproducible and with good controls. Because of the work done with interlab, we felt that we were able to identify a good positive control for our GFP-expressing plasmid. Validating that GFP signal is turned on when its promoter is unregulated, and that we can detect the fluorescence, is a great first step for our project.

For our next steps, we need to test whether the presence of ArsR will turn off GFP expression. We will do this by combining the two plasmids, BBa_K2248001 and BBa_K2248002. We will test this by measuring GFP fluorescence using the Light Cycler and the Promega S30 Extract kit. When we understand how much DNA is needed, then we will start adding arsenic to de-repress expression of GFP. Additional experiments that we may need to perform to verify that all the parts are working as expected are a protein gel or western blot, to see that ArsR is being expressed and that it’s the right size. We could also imitate the experiment performed by López-Maury, et al (2003) where they looked at the specificity of arsenic and antimony binding to the ArsR binding site using an electrophoretic mobility shift assay. This assay looks at how the mobility of proteins is slower when a protein is bound to DNA. When these experiments are worked out, we can compare this data against the combined arsenic circuit plasmid. That way, even if the complete circuit is not as efficient at sensing arsenic, we can still use the component parts and have the same effect. We will continue to refine the VK3 camera system so that it is very user-friendly and will be able to take pictures and perform an analysis. We will also improve the hardware so that the UV and white light will be combined into one piece of housing and that the user can decide which light to turn on.

The InterLab study tested 6 different Devices, with a positive and negative control. This is the order of devices from the highest fluorescence to lowest at the 6 hour time point:

Positive control
Device 2
Device 1
Device 4
LB-chloramphenicol
Device 5
Device 6
Device 3
Negative control

These results tell us that the positive control had the highest amount of fluorescence and the negative control had the least amount of fluorescence; these appear to be very good controls for the experiment.

Devices 2, 1, and 4 had fluorescence higher than the autofluorescence from the LB-chloramphenicol. These promoter/RBS combinations are good at expressing GFP.

Devices 5, 6, and 3 did not have fluorescence higher than the background levels in the culture media. We would not use these promoter/RBS combinations to look at GFP fluorescence if the GFP was being measured in cells growing in LB because GFP production appears inefficient.

The Abs600 readings gave us confidence that any lack of GFP expression was not due to dead cells. The increased Abs600 reading told us which bacteria cultures continued to grow over the duration of the experiment. The cultures with the least GFP expression actually replicated much more often than the cells expressing high levels of GFP. We think this means that the cells were able to use their energy for replicating, instead of making GFP like the other cultures. The positive control had the most GFP fluorescence but the least amount of Abs600; this supports our hypothesis that cellular energy was used to make GFP instead of replicating.

Fig. 1 and Fig. 2 show graphs of our results from the Interlab.

Team:Cadets2Vets/Results

Results

Cloning

Testing

future work

Interlab Study

Fig. 1

Fig. 2

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