Part:BBa_K2110008

inclusion body induced cell lysis system

This part consists of theCpxR responsive promoter (Part:BBa_K135000), a RBS (Part:BBa_B0034), ddpX gene (Part: BBa_K2110004), two terminators (Part:BBa_B0010, Part:BBa_B0012). The CpxR responsive promoter can response to the inclusion body and misfold protein in the periplasm. When this system coexpress with an exogenous gene like the PETase gene, the forming inclusion body will induce the expression of downstream ddpX gene and cause the cell lysis, which provides much convenience to the purification steps followed.

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]

Results

We first did a experiment to measure the cell lysis effect of ddpX gene. We simply use the IPTG inducible T7 promoter to regulate the expression of ddpX gene. We totally set four groups:
1. E.coli wildtype.
2. E.coli wildtype added IPTG.
3. E.coli with ddpX gene, no induction.
4. E.coli with ddpX gene, IPTG added as induction.
Then we continuously measure the OD₆₀₀ of the culture medium by 96-well Microplate Reader and draw the OD₆₀₀-culturing time curve by Matlab. The graph is showed below.

            Fig.2. OD600-culturing time curve of different groups of inclusion body induced cell lysis system.

From this image, we can see that in the first 5 hours, the OD₆₀₀ of each group is almost the same because of the rich nutrition and the ddpX cannot take effect immidiately. However, after 5 hours, the group with ddpX transformed and IPTG induced showed the fastest decrease among all the groups. This is because of the cell lysis effect of ddpX. Comparing the group with ddpX gene and IPTG inducted with the group with wildtype bacterial and the same amount of IPTG added we can draw the conclusion that the ddpX gene can cause cell lysis significantly. However, the data of another two groups are a little strange. We think it is because of the basic expression of ddpX can partly hydrolyze the peptidoglycan in the cell wall and provide cells with D-Ala as carbon source. This is exactly the physical use the the ddpX gene when the cell is under starvation condition. Therefore the group with ddpX gene but no induction can live longer and better when the nutritions are deficient.

Reference

Previous part BBa_K339007 acted as our reference, we improved its application by changing the mRFP gene to the ddpX gene. Click the link to see its details: https://parts.igem.org/wiki/index.php?title=Part:BBa_K339007