Part:BBa_M50560:Experience

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Applications of BBa_M50560

Initial Transformation and Amplification of FixJ and YF1 plasmids

Cloning of our plasmids was performed by Dr. Paul Vorster using Gibson assembly. Because the quantity of plasmid cloned is very small (< 10µL), we first amplified each of the plasmids by individually transforming them into chemically competent E. coli cells using a heat shock method and plating the transformed cells onto LB+Amp agar plates. We then let these plates grow overnight, picked colonies from each plate and incubated them in LB+Amp media overnight again, and then performed Miniprep DNA extraction on the bacterial cultures using a QIAGEN Miniprep Kit. After Miniprep extraction, we had ~30µL of each plasmid, and we performed NanoDrop Spectrophotometry to ensure adequate concentration and A260:280 ratio of each. We sent 10µL of each plasmid to the Stanford Protein and Nucleic Acid (PANS) facility for sequencing, and the sequencing verified that our plasmids were correct. Our system is only functional if both the FixJ and YF1 plasmids are present in the cell, so our first step was to conduct a cotransformation of both plasmids into E. Coli. When these two plasmids are expressed at the same time, mCherry should be produced in the absence of blue light and inhibited in the presence of blue light. We based our cotransformation protocol on a protocol released by the 2014 TU Eindhoven iGEM team, which calls for the exact same steps as heat shock transformation of a single plasmid, except that we introduce two plasmids instead of one13. Although the TU Eindhoven iGEM protocol does not explicitly recommend having equimolar quantities of plasmids, “Avoiding and controlling double transformation artifacts” by Goldsmith et al. in 2007 found that percent of successful double transformations increased exponentially with amount of transformed DNA, so to ensure that our plasmids were transformed at the same rate, we used equimolar quantities of each14. To calculate the volume of each plasmid needed to ensure equimolar quantities, we used the following formula: V1V2 = C2bp1C1bp2 , where V = volume (µL), C = (ng/µL), bp = number of base pairs. This equation is derived from the equations: C1V1 = C2V2, M1MW1=M2MW2(where MW = molecular weight (ng/mol)), and MW1MW2 = bp1bp2. We used 100 ng of each plasmid in the cotransformation and plated the cotransformed E. Coli onto an LB+Amp agar plate. After letting the plate incubate overnight, we observed successful growth of colonies, suggesting that at least one of the ampicillin-resistant plasmids was able to make it into cells. This cotransformation process represented one of the biggest challenges in our experimental design, as it was difficult to guarantee that both of the plasmids had been taken up by cells, and one plasmid could outcompete the other in cells because the two plasmids have the same high copy number origin of replication. According to Goldsmith et al14 and another paper titled “Plasmid incompatibility: more compatible than previously thought?” by Velappan et al. in 2007, plasmids containing the same origin of replication are generally seen as incompatible for cotransformation, meaning they yield low transformation efficiency. However, Velappan et al. also show that cotransformed plasmids with the same replication of origin are not all rapidly lost, and some are able to persist in bacteria for multiple overnight growth cycles, giving us hope initially that our cotransformation could still be successful15. Furthermore, because both plasmids individually code for ampicillin resistance, colonies that only contain one of the target plasmids still grew up on the plate, making it hard to determine which of the colonies contained both plasmids. Because of the difficulty of this step, we spent most of our project troubleshooting the cotransformation process and conducting PCR experiments to verify the presence of both plasmids. This could have been avoided by designing our plasmids to have different antibiotic resistances as well as his tags. Eventually, our final PCR showed the presence of both plasmids. See image below for successful PCR. Nevertheless, when we tested the colonies under the microscope, both the ones under dark and blue light conditions, the results were inconclusive, seeing as there was background mCherry excitation. This means that the device cannot be proven to work in the desired way, as can be seen in the images below.

Location on Stanford Campus:

Plasmid name: FixJ Organism: E. coli Device type: Sensor Glycerol stock barcode: 0133011979 Box label: BioE44 F18

Plasmid name: YF1 Organism: E. coli Device type: Sensor Glycerol stock barcode: 0133011955

Box label: BioE44 F18

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