Team:SoundBio/Project/Results
Results
Significance
Being able to spatially control the functionalization of bacterial cellulose has the potential in the field of tissue engineering to create novel biomaterials with therapeutic properties.Achievements
We successfully characterized the FixK2 promoter (BBa_K592006), showing there is little to no leaky expression.Data/Results/Analysis
Characterization of the FixK2 promoter
FixK2 promoter
To characterize the amount of leaky expression of the FixK2 promoter when it is turned off, we measured the average fluorescence of E. coli transformed with three different plasmids under different light conditions - blue light and darkness (L indicating blue light exposure and D indicating no light exposure. Our experimental plasmid (indicated by EL and ED) comprised the GFP gene under the control of the FixK2 promoter. We used a constitutively expressed GFP gene as our positive control (PL and PD) and the FixK2 promoter with no reporter gene as our negative control (NL and ND).
Our results show that the FixK2 promoter has very little leaky expression. In both light-exposure and no-light exposure tests, our experimental plasmid had fluorescence levels comparable to the negative control. Our positive control, as expected, produced significantly higher fluorescence.
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Light-controlled genetic circuits
Initially, our plan was to clone both a red light controlled circuit and a blue light-controlled circuit. Last July, we outsourced the red light circuit cloning work to Genewiz, a genomics service company, and decided to clone the blue-light circuit ourselves.
We sent Genewiz a plan to clone the red light circuit using Standard Biobrick Assembly, and we were going to use 3A Assembly to clone the Blue light-controlled circuit.
Unfortunately, after multiple cloning failures, Genewiz reported they could not successfully clone the red light circuit for us. We were also unable to successfully clone the blue light circuit due to errors in PCR and digestion/ligation steps. In general, the cloning work involved a lot of steps which would have led to a higher chance of human error. The digest reactions often did not produce our desired fragments and gel extractions were difficult with the small fragments we were working with. Our PCRs for the YF1/FixJ part did not go as planned. As shown in the diagram above, we sought to add a constitutive promoter in front of it. However, after each PCR, our gel results show a differently sized amplicon (~1 kb) when our desired product should’ve been around ~1.8 kb. We tried to troubleshoot the source of error but online primer analysis tools suggested that there was only one reasonable primer binding site which would have produced the correct sized amplicon.
Another issue we faced was the considerable length of time to run digests and ligation reactions and then transform ligated fragments into E. coli. Due to the complexity of the blue light cloning workflow, we decided to focus our time and energy on other tasks.
In August, we found another cloning service company called BIOFAB. We gave them plans to clone the red light circuit for us. The biggest issue we faced was the complexity of our circuits. The red light circuit required a special strain of EnvZ-deficient E. coli and a phycocyanobilin generator (PCB) in order to work. Oftentimes we saw deleted sections in our Gibson assembly result. As shown below, a major issue we found was cloning double terminators into our circuit. Because of the stem-loop as shown below, part of the double terminator would be deleted during Gibson assembly.
For more sequencing mishaps, check out the alignment data for our PCB Plasmid: Link
In the end, we were unable to successfully clone the red light circuit in time.
Future Implications
We hope that in the future we will be able to successfully clone the red and blue-light controlled circuit. One possible solution is to retry the Gibson assembly, but with fewer parts. This will give us more oversight of the reaction and will allow us to make modifications necessary to allow the Gibson assembly to be successful. Another possible solution is to redo the Gibson assembly and to stitch together several oligos. We may also want to consider different light-sensing pathways. With multiple optogenetic circuits, we may be able to functionalize bacterial cellulose with multiple inputs (like green light) and create a system of which we have more control over.
We sent Genewiz a plan to clone the red light circuit using Standard Biobrick Assembly, and we were going to use 3A Assembly to clone the Blue light-controlled circuit.
Unfortunately, after multiple cloning failures, Genewiz reported they could not successfully clone the red light circuit for us. We were also unable to successfully clone the blue light circuit due to errors in PCR and digestion/ligation steps. In general, the cloning work involved a lot of steps which would have led to a higher chance of human error. The digest reactions often did not produce our desired fragments and gel extractions were difficult with the small fragments we were working with. Our PCRs for the YF1/FixJ part did not go as planned. As shown in the diagram above, we sought to add a constitutive promoter in front of it. However, after each PCR, our gel results show a differently sized amplicon (~1 kb) when our desired product should’ve been around ~1.8 kb. We tried to troubleshoot the source of error but online primer analysis tools suggested that there was only one reasonable primer binding site which would have produced the correct sized amplicon.
Another issue we faced was the considerable length of time to run digests and ligation reactions and then transform ligated fragments into E. coli. Due to the complexity of the blue light cloning workflow, we decided to focus our time and energy on other tasks. In August, we found another cloning service company called BIOFAB. We gave them plans to clone the red light circuit for us. The biggest issue we faced was the complexity of our circuits. The red light circuit required a special strain of EnvZ-deficient E. coli and a phycocyanobilin generator (PCB) in order to work. Oftentimes we saw deleted sections in our Gibson assembly result. As shown below, a major issue we found was cloning double terminators into our circuit. Because of the stem-loop as shown below, part of the double terminator would be deleted during Gibson assembly.
For more sequencing mishaps, check out the alignment data for our PCB Plasmid: Link
In the end, we were unable to successfully clone the red light circuit in time.