Question

A fellow researcher is constructing a plasmid vector they are hoping to express in e. coli. As they are designing their experimental protocols they are concerned that they will not be able to identify colonies which were successfully transformed, with the correct plasmid/insertion construct. Provide a suggested mechanism or method to address their concerns. Specifically answer the following two questions and provide details on the biological mechanism underpinning the method.

a. Describe a mechanism to determine if the bacterial have successfully been transformed with the plasmid. (10 pts)

b. Describe a mechanism to determine if the bacteria, which were transformed, were transformed with the desired product (original plasmid plus insertion of interest). Note: you may design an approach to address a and b separately or possibly in a single method. (10 pts).

In the experiment above they perform a heat shock method and then plate the bacteria directly onto ampicillin containing plates. However, they get extremely weak colony growth. They are confident in the plasmid construction and transformation mechanisms, what modification to their procedure would you suggest to possibly improve colony growth? Discuss the biological foundation behind your suggestion? (8 pts)

Answer 1

Plasmids are small ectrachromosomal DNA segments that carry genes that benefit the survival of organisms.

Plasmids are introduced into cell by transformation.

Using heat shock method is basic technique in molecular biology to insert foreign plasmid into e coli. After short incubation in ice , mixture of bacteria and DNA is placed at 42 degree celcius for 45 sec called HEAT SHOCK and then placed back in ice.

Plasmid DNA (pDNA) is an alternative for immunization and gene therapy against many infectious, genetic and acquired diseases

The common host for pDNA production is the bacterium Escherichia coli. Several E. coli strains have been reported for pDNA production, such as DH5α [2–4], DH5 [5 .

E coli is used create insulin . The gene thhe that produces human insulin was added to the genes in a E. coli bacteria. ... By culturing large quantities of the modified bacteria and then killing and opening them, theinsulin could be extracted, purified and extracted .this insulin is used in diabetics.

The typical challenges for high cell-density cultivations (HCDC) of E. coli remain as obstacles for the fast and efficient production of pDNA. Among them, aerobic acetate production is an important drawback, since it causes a loss of productivity and waste of carbon source. Aerobic acetate production -known as overflow metabolism- results from an imbalance between glycolysis and tricarboxylic acids cycle .Some of the strains commonly used for pDNA production present elevated overflow metabolism, including E. coli DH5α and DH1 [9]. While the conventional way of avoiding overflow metabolism is reducing the glucose uptake in the so called FED BATCH MODE., the constant supply of glucose to the bioreactor requires additional equipment, results in a decrease of growth rate and frequently causes substrate gradients at the feeding zone in production bioreactors that trigger undesirable physiological effects . substitution of the natural glucose transport system (PTS) by a constitutively expressed galactose permease under the strong trc promoter in E. coli allows efficient growth by reducing the glucose uptake rate and consequently decreasing acetate production .

The possibility of cultivating VH33 strains and derivatives at high cell-density in batch mode is a simple and valuable alternative to fed-batch mode for the fast and efficient production of pDNA both, at early-stages of product development and at technical scale.

Improving colony growth can be done by SOC BROTH. Which is S uper O ptimal broth with C atabolite repression which is a nutrient rich medium.it is used to grow competant cells to maximize the efficacy of bacterial plamid transformation.