introduction/background Bio lab paper

o pPEM109 plasmid purification: the plasmid is the vector that will carry the target DNA (the one that is wanted to be expressed in the transfected cell, LepA/EF-4). Plasmid purification is DNA purification, normally plasmids are stored in bacteria or purified. In this case, the plasmid was purified, which can be done with lysis buffers to break bacteria, RNase treatment to degrade RNA and phenol: chloroform: isopropanol purification. The purpose of this experiment is to get the plasmid that has the target DNA (LepA/EF-4).

o GFP PCR (donor DNA) and column clean-up: green fluorescent protein (GFP) serves as a gene marker that we attached to another gene to follow it's expression. The PCR (polymerase chain reaction) will help amplify the GFP coding sequence. The PCR reaction requires the DNA to be amplified (GFP gene sequence), free nucleotides, primers, DNA polymerase, magnesium, and therefore once the reaction has finisihed a column purification of the GFP sequence will be required to separate it from all the other reactives.

o Transposition reaction: This technique is based on the transposons. Basically, helps fusion of the target DNA (LepA/EF-4) with the GFP sequence For this experiment the plasmid containing the LepA/EF-4 sequence is incubated together with the GFP sequence and transposase enzymes. This will fuse the plasmid containing LepA/EF-4 with the GFP sequence. The purpose of this is to get both gene sequences fused.

o Transformation of plasmid library into E. coli: Thermic shock is the most commonly used technique to transform E. coli bacteria, which means introducing the plasmid inside it. So, once obetained the plasmid with LepA/EF-4 fused to GFP, we add some of the plasmids to competent (made competent by previus treatment with calcium chloride) E. coli bacteria, then they are incubated at 42°C for 90 seconds and then quickly transfered to ice. This will make the E. coli cells to take up extracellular DNA, like the plasmid we added. After that, the now transformed cells can be cultured on plates with LB-ampicillin agar medium at 37°C.

o Selecting GFP positive colonies and re-streaking for single colonies; GFP is a fluorescence protein, so to detect GFP positive colonies we must look them in a dark chamber after being exposed to UV light, which will make them glow, halping us to differentiate the succesfully transformed colonies from the ones not transformed or in which the transposition reaction was not succesful and the GFP sequence was not inserted in the plasmid. Once the positive GFP colonies are identified they can be re-stroke on a new plate.

o Inoculation of GFP positive colony into LB+AMP liquid cultures minus IPTG: The Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a molecule that induces the expression of any gene followed by a lac operator sequence. All plasmids contain a coding sequence for ampicillin, so only successfully transformed bacteria will grow in a liquid culture with ampicillin in it. As we don't want to see the gene expression yet, no IPTG is added.

o Plasmid purification and sequencing: Once we have confirmed that we succesfully transposed the target gene inside a plasmid with the GFP sequence and isolated succesfully transformed cells, we need to purify the plasmid inside them as explained before (in experiment 1) and sequence the plasmid to see in which specific place was the GFP sequence inserted during the transposition reaction.

o Mapping of GFP structure onto PDB molecule model of LepA/EF-4. PDB stands for protein data bank, where we can find the function of the protein coding by our target gene, LepA/EF-4, which is to promote translation (translation factor) as well as the tridimensional structure. And the purpose of it is to determine the tridimiensional structure our target gene will have after being fused with GFP, to make sure both proteins keep their functions.