Part:BBa_K2382003

T7 promoter & Lac operator and RBS from PET-29a

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

The sequence of this BioBrick is partially originated from pET-29 a (+) Vectors, and it is composed of T7 promoter, Lac operator,and RBS.

We tried to improve this promoter sequence at BBa_K525998 by adding an lac operator and making it RFC 10 compatible, since the Lac operator from pET-29a(+) has an illegal XbaI restriction enzyme site .
The original BBa_K525998 doesn't contain Lac operator sequence, and we managed to insert one in it so it would work better with the presence of LacI protein, making its regulation function better.

Expression results

This part is a biobrick component in the expressive system(BBa_K2382006) to produce the Thioredoxing-MSMEG_5998 fusion protein(BBa K2382009), a specially designed aflatoxin degrading enzyme. In the following description, we will call this fusion protein (BBa K2382009)"Synthetic MSMEG5998", and MSMEG_5998(BBa K2382001) will be called "Australian MSMEG_5998", since the Australian MSMEG_5998 was requested from Taylor, M.C. in CSIRO .from Australia.

IPTG induction

This composite parts (BBa_K2382006) were synthesized by Allbio Life Co., Ltd and put into the standard backbone pSB1C3. First, we transformed the plasmids into E. coli BL21 (DE3) strain to express our proteins. Then IPTG was used to induce the expression system since all plasmids in our project had T7 promoter. We sonicated E. coli and did 9500 rpm and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To confirm the suitable concentration of cell supernatant, we did SDS-PAGE electrophoresis and coomassie brilliant blue staining. The results is demonstrated in the Fig. 1. After centrifuging two times, we could find a high percentage of proteins in the cell supernatant (the 13000 Su group)

Figure 1: Cell lysates were analyzed by SDS-PAGE and coomassie brilliant blue staining. 9500 T meant the initial sample obtained after sonication; 9500 P and 13000 T meant the pellet and the supernatant gotten after 9500 rpm for 20 min; 13000 P and 13000 Su meant the pellet and the supernatant obtained after 13000 rpm for 20 min. (A)(B) Samples contained Australian and synthetic MSMEG5998.

Protein purification, and dialysis

After extracting the cell lysates, we used nickel-resin column to purify our target proteins from the cell lysates because all of our proteins were tagged with 6 histidines at their C-terminal ends. After protein purification, protein dialysis with diaysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH=7.5), and 20% glycerol to remove imidazole in our purified proteins, we did SDS-PAGE gel electrophoresis to ensure our target proteins were successfully purified (Fig. 2A ). The molecular weights of these proteins are listed in the Table 1. The standard BSA proteins were used to quantify the concentration of target proteins.

Table 1: Two expressed recombinant proteins and their molecular weights are listed.

Proteins	Molecular weight
Synthetic MSMEG5998	32.4 kDa
Synthetic FGD	51.5 kDa

Figure 2B: Concentration of proteins was quantified by SDS-PAGE and standard BSA samples with 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 mg/ml. (B) Two recombinant proteins expressed by the plasmids from DNA synthesis.

Protein solubility analysis

To know whether the solubility of our two enzymes (MSMEG_5998 and FGD BBa_K2382002) increased after fusing enzymes with thioredoxin, we dissolved all cell lysates which containing pellet and supernatant and did western blot to detect the content of our target proteins. All proteins were detected by anti-6x His Tag antibody because all of them contained a 6-histidines tail when bacteria expressed them. In Fig. 3, we could find there was good expression of both Australian and synthetic MSMEG5998 in the “13000 Su” group when compared with the “13000 P” group. This result meant that most proteins were dissolved in the supernatant while few proteins deposited in the cell pellet after 13000-rpm centrifugation. However, we could not observe good solubility in both Australian and synthetic FGD because there were little or no difference between the “13000 Su” group and the “13000 P” group.

Figure 3: Cell lysates in the process of two times centrifuge were analyzed by western blot. The abbreviations of five groups were the same as Fig. 1.

Protein Expression Over Time

We transformed the plasmids that contained this part (BBa_K2382006) into competent cell E.coli BL21. After cultured overnight, measure the ABS600 and diluting the LB medium to O.D.=0.1. Then incubate at 37℃, 150 rpm until the O.D. of the samples reach 0.4 to 0.6 . Add 80ul 100mM IPTG( final concentration : 0.4mM ) to 125 ml flask and return to 37°C. From then on, after measure the O.D. values, transfer 1 ml from the induced sample and centrifuge at maximum speed for 60 seconds at RT and remove supernatant at 0, 1, 2, 3, 4, 5, 6, 7, 8 hours and 0, 0.5, 1.0, 1.5, 2.0 ,2.5 , 3.0, 3.5, 4 hours. Then we use Western Blot mehtod to amalyze the quantaty of Thioredoxin-MSMEG_5998 (Synthetic MSMEG_5998) at each time spot.

Discussion

Figure 4 : The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.

According to the data shown above, the growth curve of E.coli BL21 with Synthetic MSMEG_5998 reached the ceiling when the O.D. value was approximately at 2 while the amount of Synthetic MSMEG_5998 were still increasing.

Though the amount of Synthetic MSMEG_5998 increased consistently with time, we could not jump to conclusions that it was proper to incubate E.coli as long as possible. Another consideration was the time it would take. Just as our expected, it growed fast at the first 2.5 hours. That’s why we also chose 2.5hr after induced by IPTG when we extracted Synthetic MSMEG_5998 from total cell lysate in other experiments.

Figure 5 : The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.

Based on previous experience, if the E.coli was incubated over 4 hours, the protein that it expressed may be degraded or mis-folded, leading to malfunction. As a result, it was also an important issue for this modeling. However, because of the lack of F420, we did not have the chance to check the enzyme activity of each time spot. It was still unknown whether the titer of the Synthetic MSMEG_5998 would change or not and awaited further research.

Figure 6 : Cell lysates from E. coli BL21 with Synthetic MSMEG5998 from 0 to 8 hours and 0 to 4 hours were analyzed by Western blot. The amount of Synthetic MSMEG5998 increased consistently with time.

References

pET-29a(+) 5.4kb - GenScript https://www.genscript.com/gsfiles/vector-map/bacteria/pET-29a.pdf

BioBrick Part BBa_K525998