Synechococcus PCC 7002 engineering bacterium independent of VB12 and construction method and application of engineering bacteria
A technology of Synechococcus and engineering bacteria, applied in the field of genetic engineering, can solve the problem of not having synthetic cobalamin, and achieve the effects of reducing bacterial collection, convenient extraction and wide application.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0033] The exogenous gene was integrated into the genome of Synechococcus sp. PCC 7002 by the method of "homologous double crossover" commonly used in biology.
[0034] Homologous double crossover is to select the upstream and downstream homology arm genes at the homologous exchange gene, generally 400-3000bp is acceptable. The present invention selects the upstream and downstream homologous segment genes from the A2746 site on Synechococcus sp. The cpcBA promoter gene, the target protein expression gene, the metE gene shown in SEQ ID NO: 2, and the homology arm gene fragment downstream of A2746 shown in SEQ ID NO: 4 were connected to the pAQ1 plasmid to construct a new vector Q29. Vector Q29 was transformed into Synechococcus to realize the integration of exogenous genes through homologous double crossover.
[0035] Specifically, a method for constructing a VB12-independent Synechococcus sp. PCC 7002 engineering bacterium, the steps are as follows:
[0036] 1. Acquisition o...
Embodiment 2
[0081] Synechococcus PCC 7002 engineering bacteria and wild-type Synechococcus PCC 7002 growth contrast in liquid medium, the steps are as follows:
[0082] Wild-type Synechococcus PCC 7002 was cultivated in 5mL of A+ culture solution, and Synechococcus PCC7002 engineered bacteria were cultivated in 5mL of A+ culture solution without vitamin B12; the OD of the culture solution was measured every 2h (measured wavelength was 730nm ), the test results are as follows Figure 4 shown.
[0083] Depend on Figure 4 It can be seen that due to the expression of the metE gene in Escherichia coli, the Synechococcus PCC7002 engineering bacteria can grow in the A+ culture solution that does not contain exogenous vitamin B12, compared with the growth situation of the wild-type Synechococcus PCC7002 in the A+ culture solution, When the Synechococcus PCC7002 engineering bacteria grows in the A+ culture medium that does not contain vitamin B12, the growth rate in the logarithmic growth phase...
Embodiment 3
[0085] The verification of exogenous protein expression of the gene shown in SEQ ID NO: 5, the steps are as follows: take the wild-type Synechococcus PCC 7002 culture solution cultured in A+ culture solution for 5 days and culture it in A+ culture solution without vitamin B12 for 5 days. Each 2mL of Synechococcus PCC7002 engineered bacterium culture solution of 1 day was collected, and the precipitate was washed twice with 10mM Tris-HCl buffer solution (10mM, pH 7.6) with pH 7.6; ;Take 40μL of the resuspension solution and mix it with 2×sample buffer, put it in a boiling water bath for 15min, take 20μL of the sample and spot it on the TGX precast gel, electrophoresis at 150V for 60min; Electrophoresis was carried out at a temperature of 4°C and a voltage of 100V for 1 h in the reverse blotting method. Use the His super signal probe to mark the exogenous protein expressed by the target gene, the results are as follows Figure 5 shown.
[0086] The results showed that the expr...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More