- TANG, Chloe T.C.
- Tam, Phoebe L.F.
- Tam, Sabrina K.M.
- Tang, Mandy L.Y.
- Siu, Mona M.Y.
- Yiu, Stephanie P.T.
It is hypothesized that strong gene expression will affect the growth and viability of cells. This experiment was conducted by incorporating a strong promoter from the Anderson Family (BBa_J23101: Registry of Standard Biological Parts) with a medium ribosome binding site (BBa_I13003: Registry of Standard Biological Parts) in Escherichia coli (E.coli) to test the hypothesis. The growth rate of engineered Escherichia coli (E.coli) was then compared with that of a negative control (incorporating only BBa_I13003 in Escherichia coli (E.coli)). Quantitative results of optical density is obtained by spectrophotometer with UV light of 600nm. The results indicated that the effect of overloading cells on growth rate in Escherichia coli (E.coli) was insignificant and
further investigations are needed to test the hypothesis.
Gene expression in bacteria involves the production of messenger RNA (mRNA) from DNA template and translation of the mRNA into protein. These two processes are driven by the binding of RNA polymerase to the promoter region of the gene and the binding of transfer RNA (tRNA) to the starting codon that is nearest to the ribosome binding site (RBS) respectively. Therefore, to strengthen the expression of a foreign gene in E. coil, strong promoter and RBS are selected as two controlling factors.
Under the stress of being transcribed too often, much energy and materials are required for gene expression. It is suspected that DNA overloading will lower cell viability as energy and materials are wasted during gene expression. Also, the accumulation of proteins may perturb cellular function which in turn affects normal cell growth.Therefore, we would like to investigate the effect of overloading on cell growth and viability.
Methods and Materials
To investigate the effect of strong gene expression on cell growth and viability, a gene encodes for yellow fluorescent proteins with strong promoter and RBS were constructed and transformed into E. coli. Then, the growth rates of cells with strong promoter and RBS are compared with those without to test the hypothesis.
6 sets of similar experiments were carried out. Set 1 - Set 5 were performed as follow:
BBa_J23101 and BBa_I13003 were extracted out from iGEM kit plate and underwent transformation, followed by inoculation. Plasmids were then extracted with Mini Plus™ Plasmid DNA Extraction System. The concentration and quality of purified DNA sample was assessed by NanoDrop 2000 spectrophotometer.
Approximately 300ng of BBa_I13003 was digested with 1.5 units of XbaI (NEB, R0145L) and 1.5 units of PstI (NEB, R0140L), in 10X NEB Buffer 3 and 10X BSA. Reactions were incubated at 37˚C for 1.5 hour. DNA samples obtained from digestion were analyzed by running 1% agarose gel electrophoresis with 120V electricity for 30 minutes, which was pre-stained by Midori Green for visualization of DNA fragments. Insets were extracted out from the gel and purified using Favorgen FavorPrep™ GEL/PCR Purification Mini Kit.
Approximately 300ng of BBa_J23101 was digested with 1.5 units of SpeI-HF® (NEB, R3133L) and 1.5 units of PstI-HF® (NEB, R3140L), in 10X NEB CutSmart™ Buffer (NEB, B7204S). Reactions were incubated at 37˚C for 1.5 hour. Backbone was separated by digestion cleanup using Favorgen FavorPrep™ GEL/PCR Purification Mini Kit.
The digested BBa_J23101 and BBa_I13003 were then undergone ligation in a ratio of 1:3 with 1 unit of T4 DNA ligase, in 10X T4 ligase buffer. Reactions were incubate at 37˚C for 1 hour and underwent transformation, followed by overnight inoculation in 5ml Lysogeny broth (LB).
For Set 6, the negative control setup, was similar to Set 1 - Set 5, but without digestion of BBa_J23101 and ligation. Transformation and overnight inoculation in 5ml Lysogeny broth (LB) were still carried out.
Cell growth was monitored by optical density(OD) to measure OD600 using a spectrophotometer with UV light of 595nm. 200 μl of Set 1 - Set 6 were measured each time, using the corresponding pure medium as a blank. Set 1-6 were diluted 50-fold before measurement and then placed in shaker for incubation after each measurement. OD readings were taken every 45 minutes. After measurement, readings were converted to OD600 by multiplying OD595 with 1.83.
Results and Interpretations
As shown in Figure 1, the DNA samples for both the experiments with strong gene expression and the negative control show similar growth rate and pattern. Also, all samples showed an incomplete growth pattern.
According to J. Monod ("The Growth of Bacterial Cultures",1949), a normal cell growth pattern, in theory, could be divided into four phases: lag phase, log phase, stationary phase and death phase. According to the growth curve obtained, all the experimental and negative control samples showed an incomplete growth pattern. It is believed that the samples were in lag phase in the beginning and entered into log phase at approximately 235 minutes. As a result, it is suggested that the number of measurements taken might not be enough for showing the complete bacterial growth pattern.
In our hypothesis, it is expected that strong gene expression will affect the growth and viability of cells. As a result, the negative control sample should show a higher growth rate than that of the experimental samples. However, our results show no differences between the experimental setup with strong gene expression and the control in terms of growth rate. One possible explanation is that the promoter (BBa_J23101: Registry of Standard Biological Parts) and ribosome binding site (BBa_B0032: Registry of Standard Biological Parts) that we used in our experiments might not be strong enough and overloading of cells were not generated. Therefore, their growth rates show similar pattern. Another possible reason is that overloading of cells might not have significant effects of its growth rate. Hence, it is hard to make concrete conclusion simply base on the results obtained. Further investigations are needed to test the hypothesis.
The negative control setup in our experiment was not the best option. Our experiment incorporated a strong promoter and a RBS to investigate on whether overloading of cells will affect cell growth and viability. The best control setup should only contain the coding sequence of yellow fluorescent protein and the double terminators (BBa_E0130: Registry of Standard Biological Parts). However,as the desired part was not delivered by the iGEM headquarters via the distribution kit and due to time constrain, we used the part that contained the RBS, coding sequence of yellow fluorescent protein and the double terminators (BBa_I13003: Registry of Standard Biological Parts) as the negative control. One way of improving the control setup would be making it by our own. This is done by incorporating the coding sequence of yellow fluorescent protein (BBa_E0030: Registry of Standard Biological Parts) with the double terminators (BBa_B0015: Registry of Standard Biological Parts).
Both the experimental cells and the negative control showed a similar growth pattern and rate. It is concluded that cells with our synthesized promoter and ribosome binding site have insignificant effect on its growth rate. Further investigations are needed to test the hypothesis.