Science Homework Help

i attached all the information needed below

this is my introduction

Introduction-

When the process of DNA cloning was discovered it revolutionized the study of molecular biology. It enabled scientists to select a specific part of a DNA sequence and to produce it in an unlimited amount by placing the DNA fragment within a plasmid shuttle, or vector. DNA cloning has allowed scientists to amplify DNA which has a variety of applications in genetics. This includes being able to study its sequence to better understand how DNA codes for traits, it gives scientists the opportunity to manipulate and engineer the DNA, and to use it for expression and generation of protein (Sadava et al., 2009). For example, the discovery of DNA cloning allowed the mass production of the hormone insulin for diabetes which has saved countless lives (Goeddel, 1979). The ability to generate protein has also proved to be particularly useful and can be used to produce antibiotics such as the bactericidal antibiotic gentamicin, which is the focus of this paper.

There were several discoveries which enabled us to understand DNA cloning as we do now. In 1944, Avery, Macleod, and McCarty demonstrated gene transfer was possible using isolated DNA from the bacteria S. pneumoniae, this showed it is possible to obtain fragments of DNA. In 1968 by Arber and Linn, discovered an enzyme which could selectively cut exogenous DNA, which provided the mechanism to incorporate a DNA fragment into a plasmid in order to amplify it. They called this enzyme a restriction factor (Quail, 2010).

Restriction enzymes are crucial in DNA cloning as they are needed to digest the DNA sample and plasmid vector. The same enzyme is used for the plasmid and DNA in order for the restriction sites to match and ensure successful ligation. This is achieved by finding a palindrome sequence (sections of DNA exhibiting two-fold symmetry) for the enzyme (Lodish et al., 2000). To increase the chances of ligation restriction enzymes usually leave overhangs, for example Bam HI leaves a 5’ overhang and Kpnl leaves a 3’ overhang (Quail, 2001). In this experiment, a type II restriction enzyme called HindIII was used (Lab Manual, 2020). Type II enzymes are distinguished from other types of restriction enzymes by the ability to cleave DNA at specific locations within the restriction site. Then, DNA can combine with the plasmid and the enzyme DNA ligase joins the DNA and plasmid together in a condensation reaction creating phosphodiester bonds and water as a by-product. To improve the chances of ligation alkaline phosphate is used to remove 5’ phosphate groups from DNA to prevent self-ligation.

Once the plasmid is inserted into E. coil, the bacterium must be grown under restrictive conditions using an antibiotic growth medium, as the chances of successful recombination is small. Wild-type E.Coli cultures do not contain the gentamicin resistance gene. Therefore, to select for the recombinant plasmid the E. coil bacteria can be allowed to grow for around an hour in a growth-rich medium, to allow the gene for antibiotic resistance of gentamicin to be replicated. Then, when the E. coil is plated on agar with gentamicin, only those that have successfully taken up the ligated vector will survive.

The purpose of this study was to insert a DNA sequence coding for gentamicin into the bacterium E. Coli by using the Hind III restriction enzyme to digest the plasmid creating a site for the DNA, then running the fragments of the cut plasmid on a agarose gel to sequence the correct plasmid. Gel electrophoresis was used to determine the digested products of the restriction enzyme. Overall this study outlines the full process of cloning DNA and inserting into a bacterium.

i will provide all the results and changes that happened in this lab

for material and method rewrite this in another way

All experimental procedures involving the amplification and purification of the plasmid vector, digestion of restriction enzyme, ligation, purification of ligation, gel electrophoresis and lastly electroporation were conducted as written on pages 37 through 49 in exercise 3, “Basic Cloning Methods,” of the Laboratory Manual. In step 5 of part B, the addition of N4 solution appeared to have somewhat of a cloudy mixture.

our group did not have growth on the plate because the machine for electroporation did not beep which cased out plate to have no growth

All experimental procedures involving the transformant plasmid vectors, gel electrophoresis, sequencing of insert were conducted as written on pages 51 through 59 in exercise 4, “Cloning Verification,” of the Laboratory Manual.

The experimental procedure to use the ApE software in order to genetically map the plasmid resistant to gentamicin was conducted as written on pages 61 through 62 in exercise 5, “Bioinformatics,” of the Laboratory Manual.

pictures are provided for results

i will provide example from other student to make it clear