Difference between revisions of "Part:BBa K1602055"

Latest revision as of 18:18, 21 October 2021

araCpBad-GFP

Contribution:Team UGM_Indonesia 2021 Characterization

Group: Universitas Gadjah Mada / Contributors: Yustika Sari, Setianing Wikanthi

Summary Result

We tested BBa_K1602055 encoding AraC-pBAD promoter into pSB1C3 vector in two E. coli expression system: BL21(DE3) and DH5-alpha. Our data showed that the highest GF-Protein expression was observed in E. coli BL21(DE3) due to its specialization for heterologous protein expression.

Aims

The construction of BBa_K1602055 encoding AraC-pBAD promoter into pSB1C3 vector in E. coli expression system. In this study, the level of its expression was compared within two strains of E. coli, BL21(DE3) and DH5-alpha, to investigate the expression profile. Several putative transformant colonies were selected on LB agar medium supplemented with Chloramphenicol antibiotic and verified by colony PCR using VF2 VR primers.

Experiments

Several putative transformant colonies were selected on LB agar medium supplemented with Chloramphenicol and verified by colony PCR using VF2 VR primers. Overnight cultures from positive clones were used as inoculum (1% v/v) to make the working cultures. Additionally, E. coli strains, BL21(DE3) and DH5-alpha, without carrying targeted genes were cultivated as the control. The working cultures (LB broth supplemented with chloramphenicol) were incubated at 37oC and 200 rpm agitation. When the OD reached 0.1, 2mM L-arabinose was added to the medium as the inducer. For the control, we use a non-transformed culture of E. coli BL21(DE3) and DH5-alpha. The cell growth is measured using spectrophotometer OD 600 and the fluorescence intensity is measured using Spectro-fluorophotometer with excitation and emission at 504 nm and 516 nm wavelength respectively.

Results

Fig 1, Comparison of the growth curve between (a) the transformant (BL18P) and control (BLCTRL) in E. coli strain BL21(DE3), and (b) transformant (DH18P) and control (DHCTRL) in E. coli strain DH5-alpha. The measurement carried out by spectrophotometer at 600 nm.

All the E. coli transformant strain BL21(DE3) and DH5-alpha showed exponential growth lower growth compared to control. Thus, it indicated that the cells were in fact be growing slowly. This result suggested that higher metabolic burden of transformed strain. The foreign genetic part (BBa_K1602055) expression demands more cellular resources and energy, which competes with native host metabolism [1]. Hence, the host cells may suffer from delayed growth at the begining. The lag phase of E. coli BL21(DE3) clones from both transformant and control showed a shorter lag phase compared to the DH5-alpha. It indicated that BL21(DE3) has a better metabolic profile and less sensitive to metabolic stress [2].

Fig 2. Result of fluorescence measurement: (a) BL21(DE3) transformed and the control, (b) DH5-alpha transformed and the control.

The fluorescence protein was expressed in the transformant cells from both E. coli strains after the induction of L-arabinose. Both BL21(DE3) and DH5-alpha clones were induced after approximately three hours cultivation. As we expected, the fluorescence excitation depicted that expression of GFP was significantly higher in BL21(DE3) clones compared to DH5-alpha clones. It revealed that the DH5-alpha strain is not specialized for heterologous protein expression. After 6 hours culture, the remaining cells were harvested by centrifugation at 6,800 rpm for 3 minutes and observed under the UV transillumination. The fluorescence of the DH5-alpha clones was very low to be seen, the GFP expression in BL21(DE3) showed remarkably high. It can be noticed by the greenish luminescence of the cell pellet.

Fig 3. GFP luminescence under the UV light of each cell pellet for DH5-alpha (left) and BL21(DE3) (right) after centrifugation.

Team UAlberta 2018 Improvement

Composite part consisting of an araC-regulated pBAD-promoter (BBa_K808000) cloned upstream of a ribosome binding site (BBa_B0034) and GFP (BBa_E0040). Because of the araC-regulated pBAD-promoter production of the GFP is induced by the addition of arabniose. In the presence of glucose araCpBAD shows a very low basal expression level and therefore prevents GFP-expression.

The 2018 UAlberta iGEM team assembled an improved version of this part - BBa_K2779912. Our improved part allows for the efficient capture of GFP by fusion to a 6xHis tag at the N-terminus, as well as allowing for easy replacement of GFP for a different gene OR the tagging of a protein of interest with both His tag and GFP.

Functional Parameters

In order to assemble the final riboregulator the parts RRL3G (BBa_K1602045) and RRK3 (BBa_K1602046) on two seperate plasmids (pSB1C3 and pSB1A2) were co-transformed into E.coli(Top10). Positive transformants were selected by using two antibiotics, Chloramphenicol and Ampicillin, and verified via colony-PCR.

As controls served a culture of TOP10 without plasmid, one transformed with araCpBad-GFP (BBa_K1602055) as positive control and one transformed only with the cis-repressed part of the riboregulator (RRL3G - BBa_K1602045) as negative control. All four cultures were grown in LB-medium with the respective antibiotics containing 20mM glucose at 37°C over night. Afterwards 10µl of each culture were inoculated in two seperate flasks of LB-medium (with the respective antibiotics), one containing 20mM Glucose, the other one 2mM arabinose. After 16 hours of incubation at 37°C 1 ml of each culture was pelleted by centrifugation and resuspended in PBS for subsequent FACS-measurements.

The positive control (Fig.2 B) showed a significant difference in the detected fluorescence-levels between the culture grown with glucose and the culture grown with arabinose indicating that the addition of 20mM glucose to the medium is sufficient to repress GFP-expression through the araC-regulated pBAD-promoter. Surprisingly we were not able to detect the same difference in the cultures containing the assembled riboregulator (Fig.2 D). The measured fluorescence for the induced culture grown with arabinose was the same as for the culture grown with glucose. Furthermore was the detected GFP-Signal very simmilar to the results of the negativ controls (Fig.2 A+C) what leads to the conclusion that no GFP was expressed in the culture containing the riboregulator at all.

We have to assume that the interaction between the two parts of the riboregulator does not happen as anticipated, leaving the riboregulator-system constantly "locked" and preventing GFP expression, even after induction.

One possible reason for the malfunction of the riboregulator could be the fact that both parts were located on seperate plasmids which were co-transformed into the cells. It is possible that this results in an unfavorable situation for the bacteria to produce enough of both parts necessary for the riboregulator to work.

To further investigate this hypothesis we cloned both parts of the riboregulator next to each other on one plasmid resulting in the BioBrick RRK3-RRL3G (BBa_K1602047) but we were not able to repeat the experiment so far due to time constraints.

Sequence and Features