Part:BBa_K3771075

Ptrc-CDO1

Description

P_trc-cdo1 is a composite part consisting of the trc promoter and the cdo1 sequences. This part was used in in vivo testing of taurine production. The sequence for cdo1 and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Biology

trc promoter constitutively facilitates the expression of CDO1 enzyme.

Fig. 1. Taurine production pathway

Usage

CDO1 was used in in vivo testing of taurine production. The sequence for CDO1 enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Characterization

Fig. 2. Confirmation of cdo1 fragment by PCR. M: Marker; Lane 1: cdo1 (603 bp)

Fig. 3. Confirmation of pSB4KI-P_trc-cdo1 by digestion. M: Marker; Lane 1~3: Different colonies of pSB4KI-P_trc-cdo1 (5470 bp)

Fig. 4. Transformation / CDO1 in DH5α

Fig. 5. Confirmation of protein expression of CDO1. M: Marker; Lane1: CDO1 in DH5α without induction; Lane2: CDO1 in DH5α with induction (~22 kDa)

In Vivo Test 1: Choosing a Growth Medium Since the intestine is not as nutrient-rich as LB (lysogeny broth), we chose to test taurine production in both LB and M9 (minimal salts) mediums. M9 consists of fewer nutrients than LB and more closely mimics the nutrient composition of the human intestine. First, P_trc-cdo1 (BBa_K3771075), P_lacI-csad (BBa_K3771076), and P_trc-cs (BBa_K3771078) were transformed into DH5α strain. In another sample, only P_trc-cdo1 and P_trc-csad (BBa_K3771077) were transformed. The two samples were each grown in LB with IPTG inducer (LB+), M9 with IPTG inducer (M9+) , and M9 without IPTG inducer (M9-) mediums. The samples’ respective growth curves are shown in figure 12 and 13. Interestingly, the sample with all three enzymes (Ptrc-cdo1 +PlacI-csad +Ptrc-cs) had a lower growth rate in M9+ medium than in M9- medium. In the sample with two enzymes (Ptrc-cdo1+Ptrc-csad), M9 medium with or without an inducer added produced similar growth curves. Both samples grew best in LB (Fig. 12, 13)

Fig. 6. Growth curve of P_trc-cdo1 +P_lacI-csad +P_trc-cs in different growth mediums.

Fig. 7. Growth curve of P_trc-cdo1+P_trc-csad in different growth mediums.

References

1. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093
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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 534
  
• 1000
  COMPATIBLE WITH RFC[1000]