Team:Sriwijaya/Characterization

as

CHARACTERIZATION

   LacI or glucose can repress and inhibit the expression of GFP meanwhile lactose and IPTG can activate it. The lac promoter with GFP gene downstream can simply be characterized by induction and addition of glucose and IPTG to activate or deactivate the GFP system.

   We prefer to characterize the Part:BBa_K741002 because of the accessibility of the materials. We aim to demonstrate the effect of glucose or IPTG addition to GFP expression. The strains used in this characterization is TOP10, BL21, and DH5alpha E. coli strains.

  1. Competent cell preparation.
  2. Transformation of the Part:BBa_K741002 into DH5a, BL21, and TOP10 strain.
  3. Transfer of a single colony to 2 ml tryptic soy broth medium with 50 µg/ml of chloramphenicol. Incubation at 37℃ for 60 minutes.
  4. Addition of 0, 500, 800, 1000 mM of glucose and 1000 mM of IPTG.
  5. Transfer of 100 µL from each group to each well. Each sample is transferred to 2 wells.
Figure 1

Figure 1. Sample groups

After the induction of glucose and IPTG, the optical density (OD) and GFP fluorescence is measured using plate reader.

  1. Measurement of optical density using plate reader (wavelength= 490 nm).
  2. Incubation at 37℃ for 15 minutes.
  3. Measurement of optical density using plate reader (wavelength= 490 nm).
  4. Incubation at 37℃ for 15 minutes.
  5. Measurement of optical density using plate reader (wavelength= 490 nm).
  6. Incubation at 37℃ for 15 minutes.
  7. Measurement of optical density using plate reader (wavelength= 490 nm).

   Furthermore, the inducted bacteria are harvested and put into lysis mode via alkaline solution until denaturation occurred. The addition of neutralizing buffer began the process of precipitation and left the bacterial DNA plasmids in the solution. The plasmids in the solution is precipitated and spun in order to purify and separate the nucleic acid from the solution. The plasmid is isolated and ran through gel electrophoresis to analyse the remaining genetic fragments.

RESULTS

   We follow the protocol and make sure that our fluorescence standard curve are in line with the protocol using the iGEM measurement kit. With the curves shown below.

Figure 2a Figure 2b

Figure 2. Fluorescence Standard Curve.
imgDesc">To convert OD600 to µM Fluorescein

Figure 3a Figure 3b

Figure 3. (a) single colony successfully expressing GFP, (b) PSB1C3 expressing RFP (negative control)

   Three groups are set throughout this experiment with group A containing DH5a strain with glucose and IPTG induction with varying concentrations spread out through 5 subgroups, group B containing TOP10 strain, group C containing BL21 strain. Well A1-2, B1-2, C1-2 are set as negative control without the addition of any glucose (0 mM glucose). Well A11-12, B11-12, C11-12 are blanks. See figure 1.

Figure 4

Figure 4. TSB medium with 3 different concentration of glucose: 0 mM, 500 mM, 800 mM, 1000 mM; 1000 mM IPTG, and TSB medium without glucose nor IPTG as negative control.

   Each well contains 100 µL of solution. The optical density of each well is measured using plate reader under 490 nm wavelength. Results of each group are shown in bars and tables below. Optical Density is repeatly measured after 15 minutes, 30 minutes, and 45 minutes of incubation (37°C) and ran through the plate reader again consecutively for the until the 4th time.

Figure 5 Figure 5 Table

Figure 5. µM Fluorescein/OD at 0 min, 15 min, 30 min, and 45 min of incubation of cell cultures originated from DH5a colony.

Figure 6 Figure 6 Table

Figure 6. µM Fluorescein/OD at 0 min, 15 min, 30 min, and 45 min of incubation of cell cultures originated from DH5a colony.

Figure 6 Figure 6 Table

Figure 7. µM Fluorescein/OD at 0 min, 15 min, 30 min, and 45 min of incubation of cell cultures originated from DH5a colony.

Figure 8

Figure 8. Electrophoresis result on a 2% TAE agarose gel.

   A control mechanism of plac in response to glucose increases the production of β-galactosidase in the absence of glucose. Cyclic adenosine monophosphate (cAMP) is inversely proportional to that of glucose. It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site, which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase, enabling the cell to hydrolyse lactose, release galactose and glucose, and activates the GFP system. Meanwhile in response to presence of glucose, the production of β-galactosidase decreases and in the following chain of events blocks the binding of CAP-cAMP to the DNA and therefore lactose hydrolysis doesn’t occur and the GFP system is deactivated. On the other hand, IPTG works almost analogously to galactocydes therefore the induction of IPTG represses the lactose hydrolysis thus the GFP system is activated.

   In accordance with the theory, the additon of glucose deactivated the GFP system in dose-dependent manner. The Part:BBa_K741002 designer stated that the leak expression of plac is quite high. E. coli can not express enough lacI to repress the expression of plac. Even without the existence of IPTG, GFP is expressed. This is why the control group (0 mM glucose) produced the highest value of Fluorescein/OD in our experiment.

   On the other hand, our results suggest that the addition of 1000mM IPTG doesn’t activate the GFP system. On the contrary, the Fluorescein/OD produced is even lower than 0 mM glucose and 1000 mM glucose group. This might be because of the limitation of our research such as the scarcity of the materials needed for this experiment and also limited funds, so that we have to use our lab's IPTG which might have been damaged due to improper and/or prolonged storage.

   In order to confirm that the Part:BBa_K741002 is indeed transformed to each strain, we isolated the plasmid and ran it on 2% TAE agarose gel electrophoresis along with 100 bp DNA ladder and PSB1C3 plasmid as positive control for 1 hour. The DNA band is not parallel to any of the marker. That’s the only DNA ladder we have in our lab (due to our lack of fund) with 1517 bp as the highest molecular weight. Whereas, the pSB1C3 is 2070 bp and the pSB1C3-BBa_K741002 is 3153 bp. We concluded that all three of our bacteria strains contain the Part:BBa_K741002. The BL21 and TOP10 band is slightly darker. We have considered the possibility that the plasmid concentration in both strains might be lower. Hence, the isolated DNA is less or there may be another mechanism we haven’t figured out.

REFERENCES

[1]Ramey, W.D. 2002. Experiment A4a – Effect of glucose, lactose and sucrose on the induction β-galactosidase. pp. 1-6. In W.D. Ramey (ed.), Microbiology 421 manual of experimental microbiology, The University of British Columbia, 2002.

[2]Marbach, A., & Bettenbrock, K. 2012. lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA. Journal Of Biotechnology, 157(1), 82-88.