stevendort I'm confused about beta galactosidase; is it added into the plasmid before the plasmid is put into the bacteria? or is it put in after the bacteria are tested for if they are transformed

      prettypink1881 Sorry for the late reply.

      But the beta galactosidase gene is added after the bacteria with the recombinant plasmids have been identified.
      Essentially, once the bacteria are exposed to ampicillin and then tetracycline, then the recombinant bacteria have been identified, these bacteria are opened up again and there plasmids are removed once again. EcoRI (restriction enzyme) cuts upstream the insulin gene, and EcoRI also cuts the beta galactosidase gene. The gene and plasmid are mixed together and a new recombinant plasmid will form as they have complementary overhangs created by EcoRI. The 'recombinant plasmid with the beta galactosidase gene' contain the tetracycline, ampicillin and beta galactosidase gene. Originally, the recombinant plasmid only had the tetracycline and ampicillin gene.

          In the VCAA exam, What would I write if a question ever asked us to explain the steps involved in gene cloning and the expression of human insulin in bacteria (4 marks)?
          Re: 2021 VCAA Biology exam question 10c answer

            HKS
            Preface: The question talks about insulin gene cloning in 2020, meaning you would clone the whole insulin gene at once. If it talks about how they would do it in the 1980s (unlikely) you would have the say they cloned the insulin A and insulin B chain separately.

            Not going to admit, this question is a bit rubbish as they say 'cloned and expressed' in bacteria, but the examiners report only talks about cloning the insulin gene. Nowhere does it talk about the insulin gene being expressed.

            I'd personally write the whole process out as I wouldn't want to risk the marks.
            But, if I were to attempt to condense this information, I'd write the following:.

            Reverse transcriptase synthesises synthetic cDNA molecule of insulin with no introns.
            Restriction endonuclease cut sticky ends upstream and downstream insulin gene and same endonuclease cut sticky ends on plasmid.
            Insulin gene mixed with plasmid, complementary overhangs means insulin gene may anneal to plasmid. DNA ligase repairs phosphodiester bonds along sugar phosphate backbone.
            Plasmids are delivered into bacteria.
            Bacteria are grown on antibiotics, and bacteria with recombinant plasmid are identified.
            Bacteria transcribe and translate insulin gene, producing insulin protein.
            Insulin protein is isolated from bacteria.

            (this is still long sorry)
            If you have time, try write the whole process, if you are familiar with it, on the extended response page. But honestly, I think this question is incredibly poorly worded, the examiners report does not even answer their question.

              would i write about beta-galactosidase becuase its allows a fusion protein to be produced which is then purified to isolate the insulin A and B chains?

                HKS If you have the time to add it yes, but the specific question states that it is in the 2020 context, thus you would not talk about insulin A and B, just insulin as a whole.

                  but then why does vcaa say on their FAQs doc that:
                  Students should understand that one of the main methods for the commercial production of human insulin involves the insertion of genes for two different insulin polypeptides into two different plasmids in two separate bacteria. The insulin genes are inserted next to a gene for β-galactosidase protein, which allows for detection of successful gene insertion. Expression of each gene from the two bacteria allows a functional fusion protein to be produced. Students should understand that introns need to be removed prior to inserting the relevant insulin gene. Once the genes are expressed and the fusion proteins are produced by each bacteria, these fusion proteins are then purified, and the insulin polypeptides are removed and then combined together to produce functional insulin.

                    HKS This is the traditional way of manufacturing human insulin in the 1980s. If the question does not specify a year, like 2020, you would talk about insulin A and B. It was just in this specific context of how they do it in 2020, in which they do it all in one bacteria.

                        stevendort thank you!
                        also, in the recombinant bacteria being identified part, is it the tetracycline what makes the bacteria blue? or is the blue part beta galactosidase in the transform part?

                            prettypink1881 Hi,
                            So the tetracycline resistance gene gives the bacteria resistance to the antibiotic tetracycline, allowing the bacteria to grow in an environment with tetracycline. The beta-galactosidase gene, which synthesises beta-galactosidase, once hydrolysed in a substance called X-gal, breaks that beta-galactosidase into galactosidase and indole. This indole compound is what is responsible for bacteriaeria turning blue.

                                Write a Reply...