Glowing Bacteria Lab

Bio 11H lab

Introduction: In this lab we are going to make protein glow by bacterial transformation. Bacterial transformation is done by inserting DNA into bacteria where it will replicate. It uses an inducible promoter to regulate gene expression. We used a special RNA Polymerase (T7) which is controlled by the LAC promoter. Without the T7 polymerase glowing proteins could not be expressed. Normally the Lac repressor is activated making the glowing proteins non expressible. But by adding IPTG the repressor is inactivated. The glowing proteins information is then transcribed and then translated in glowing proteins. The Plasmid is also another important component in this lab. Plasmids can introduce foreign DNA into a cell and also carry its native DNA. The Plasmid is make up of four main components, the Origin of Replication, Multiple Cloning site, Promoter, and the Selectable marker. The Origin of Replication is a DNA sequence which the bacteria can start copying the plasmid. The Multiple cloning site is a very small but important DNA sequence; it contains many unique restriction enzymes that allows scientists to manipulate. The Promoter is a DNA sequence is located just a bit upstream of the coding sequence. It basically tells the RNA polymerase where to attach. The selectable marker is a gene that can code for specific antibiotic resistance. When using these genes, only cells with it can grow and thrive. This can allow easy identification of what cells transformed. GFP and BFP stands for Green Fluorescent protein and Blue fluorescent protein. GFP was first extracted from Jellyfish Aequrea Victoria in the 1970’s. Once the DNA sequence was identified they could use genetic engineering to introduce fluorescent proteins into other organisms. BFP is GFP with two amino acid substitutions. They are both very small in size, approximately 27 kilodaltons. GFP emits light by absorbing blue light and emitting green light, while BFP absorbs violet light and emits blue light. By adding in these glowing proteins into the plasmid we should be able to transform the bacteria.

Objective: 

Investigate bacterial transformation and also gene expression

Hypothesis: 

The –DNA will grow some bacteria but not glow, the –DNA +Amp will not grow, the +DNA +Amp will grow some glowing bacteria, and the +DNA +Amp +IPTG will glow a lot of glowing bacteria.

Materials and Procedure: 

See Bio 11H handout.

Data, Observations and Calculations: 

The plates the next day have grown bacteria colonies and our lab is successful. We placed our plates under UV light and we can see that our hypothesis was accurate and we got our bacteria colonies to grow.