You are designing a plasmid of DNA that encodes a gene that you want expressed in a cancer cell. When the plasmid is completed, it will be used to synthesize artificial mRNA using a standard ex vivo synthesis kit. While designing the DNA, you decide to encode the poly-A tail because you know that Poly-A tails are required for effective eukaryotic cell translation of mRNA. Once your plasmid is designed, you put it into bacteria to amplify the plasmid to give you enough copies for your ex vivo synthesis, but to your dismay, you can't seem to get your plasmid to grow in the bacteria. You use positive and negative controls to ensure that your techniques are good, and both of your controls are working fine. What could possibly be wrong with your plasmid?
Group of answer choices:
Poly-A tails are composed of a long string of A-T bonds, which are very weak, so when you put your plasmid into the bacteria, that section of the plasmid easily fragments during synthesis.
Your plasmid doesn't have the proper methylation patterns to support your gene of interest. You will need to add specific sites into your gene to encourage proper hemimethylation.
The bacteria that you are trying to grow the plasmid in only has machinery to support the synthesis of eukaryotic linear chromosomes, so the plasmid won't grow because it is circular in nature.
Your plasmid doesn't contain a codon for the cap of the mRNA, so while you have the poly-A tail, you don't have a cap to accompany it.