Question

Need help filling in the chart and answering the questions that go along with it. I have added the procedure and the instructions as well as the "results" that are supposed to be used to fill in the chart. Thank you!

Answer 1

Plate	Inoculum	Amount of growth	Appearance in normal light	Appearance in UV light
LB	E.Coli - DNA	Confluent (mat) growth of cells	Normal growth observed	No Fluorescence seen
LB/AMP	E.Coli - DNA	NO growth as wild type is AMP .sensitive	NO Growth observed	No Fluorescence seen
LB/AMP	E.Coli + DNA	Growth of transformants which have only AMP resistance gene but is less due absence of non utilisation of Arabinose. ,	Transformants growth observed	No Fluorescence seen as Arabinose is not present in medium hence GFP is not expressed
LB/AMP/ARA	E.Coli + DNA	Growth of transformants observed which have AMP resistance and also Arabinose utilisation genes	Transformants growth observed	Fluorescence seen as Arabinose is present in medium hence GFP is expressed

Answer 1: A plasmid named pGLO carrying green fluorescence proteins (GFP) gene was used as a vector to transform the recipient E.Coli cells.

Answer 2: bacterial transformation is a laboratory method of DNA transfer in to recipient cells. The addition of calcium chloride increases the ability of bacterial cells to take up plasmid DNA.The addition of calcium chloride to a cell suspension helps binding of plasmid DNA to lipopolysaccharide (LPS) of recipient bacterial cell wall, this happens as positively charged calcium chloride attracts both plasmids DNA and LPS of recipient cell, both of which are negatively charged. Thus presence of calcium chloride makes recipient cell competent and if at this point the recipient cell is given a heat shock the the recipient cell membrane will become more permeable and allow uptake of external DNA ( plasmid DNA).

If heat shock is not given then the transformation capacity is drastically reduced thus both calcium chloride and heat shock play a major role in bacterial transformation.

Answer 3: The ampicilin resistance and green fluorescence are indication of transformation and is checked by observing the growth of organisms on different media.

In this case (E.Coli - DNA)   the growth on LB medium without ampicilin shows that organisms are capable of growing on LB. No growth on medium with ampicilin shows that the organism is sensitive to ampicilin.

In case of (E.Coli + DNA) the presence of growth of the organism on LB medium with ampicilin indicates that the recipient cell has been transformed completely , but due to absence of Arabinose in the medium , GFP gene is not expressed and hence no fluorescence is seen under UV.

In case of (E.Coli + DNA) the presence of growth of the organism on LB medium with ampicilin + Arabinose indicates that the recipient cell has been transformed completely, and due to presence of Arabinose in the medium, GFP gene is expressed and hence no fluorescence is seen under UV light.