Question

You are tasked to clone the human actin gene (blue) from a cDNA clone that you obtained from a collaborator into the cloning vector termed puc119 using the Hindtil restriction enzyme. The 4 blue arrows show the Hind II recognition sequence on the Actin cDNA clone. Pin MCS MB1 ani MAIG PUC119 3162 bps CDNA Clone Figure 2: a. pUC119 cloning vector; b. cDNA clonc obtained from your collaborator showing Hind III restriction sites (arrows) e. How would you confirm that the cloning vector contains the complete gene fragment. Please explain fully how you would differentiate between bacterial cells containing the cloning vector with (a) no insert (b) partial actin gene frangment (C) complete actin gene fragment. (15 points)

This is all I was given. Pls help.

Answer 1

e. In order to confirm if the cloning vector contains the complete gene fragment, we need to culture the bacterial cell containing the vector in suitable medium and obtain the protein product. Now we can isolate the protein product using gel electrophoresis and know if actin protein is produced by the bacterial cell. If actin protein is isolated, then the cloning vector contains the complete gene fragment.

OR

In order to confirm if the cloning vector contains the complete gene fragment, we can treat it with Hind III restriction enzyme and cleave the DNA into two parts - one containing the insert and another containing the rest of the DNA. Now we can subject these two parts to gel electrophoresis and measure the sizes of these fragments. We can now match the size of the "insert" band with the already known size of the insert to know if the insert has been completely inserted into the cloning vector.

Differentiation between bacterial cells :

Bacterial cells with complete actin gene fragment can be differentiated from cells with no insert and with partial insert by detecting the expression of actin gene and production of Actin protein which can be isolated and verified through gel electrophoresis.

Bacterial cells with no insert do not produce the Actin protein and bacterial cells with partial insert may produce no Actin protein or produce truncated Actin proteins. However these cannot be differentiated by detecting their gene expression. It is rather best to subject both types of cells to restriction enzyme treatment followed by subsequent gel electrophoresis to find out the sizes of the fragments. If the bacterial cells do not contain any insert, no "insert" band will be observed. If the bacterial cells contain partial inserts, then an insert band of lower size than expected will be observed.

OR

The bacterial cells with no insert and partial inserts can be differentiated by subjecting them to Sanger sequencing and finding out the nucleotides present in both.

Bacteria