Team:SYSU-China/project/cyclecontrol

 

Introduction

We can regulate bacteria cell cycle by controlling DNA replication, RNA transcription, or protein translation, but regulation of DNA replication initiation is promptest and do the less harm to basic metabolism.

DNA replication in bacteria is controlled at the level of initiation. Bidirectional chromosomal replication from the unique replication origin, oriC, toward the terminus, terC, requires a constant time at a given temperature and assisted by lots of proteins.[1]

Method

Plasmid Construction

Gene iciA, hda, seqA, cspD was cloned from E.coli strain BL21, and link to RFP. We test the function of this four genes in vector pUC18 respectively.

Figure 1. gel electrophoresis of 4 cycle regulative genes (A: PCR 4 genes from genomic DNA of BL21 strain with self-designed primers; B: connection 4 genes with RFP respectively then link to pUC18 vector.)

 

Growth

1. Transform the vectors into E.coli strain DH5αas to amplify vectors and measure the Specific parameters of cycle delay. Shake the bacteria in 50mL LB liquid in 37℃, 260rpm. Extract 100ul Bacteria Solution and store on 96-well plate in 4℃ every two hours for protein accumulation test. Extract 100ul Bacteria Solution and measure bacteria concentration through OD600 every hour. *IPTG was added in OD600=0.2, final concentration=0.5mL

2. Transform the vectors into E.coli strain GE in whose chromosome GFP gene was integrated. Shake the bacteria in 50mL lactose liquid media in 37℃, 260rpm. Extract triplicate of 100ul Bacteria Solution and store on 96-well plate in 4℃ every two hours for measuring protein production, energy consumption, and metabolic efficiency. *IPTG was added in OD600=0.2, final concentration=0.5mL

Figure 2. E.coli grow in shaker (IPTG induced, 37℃)

Result

Bacteria Cell Growth Rate

Figure 3. Curve of bacteria concentration in OD600 by hours(time range from 0 to 12 hour. A:iciA, B:hda, C:seqA, D:cspD)

Compare the growth curve of 4 cycle repression genes transformed bacteria with control group, we can see a conspicuous difference between them and estimate the cycle repression rate(r) by modeling. The smooth period is quite close, which means that there is proteins have no significant effect on max environmental capacity.

Cell Morphology Observation

Figure 4. photograph of E.coli under microscope(A and C are capture in 510nm light; B and D are capture in white light)

We can find that cells with cycle repressive gene transformed (img3-D) are much larger and longer(2 to 3 times) than control group cells (img3-B)

Metabolic Efficiency

Figure 5. protein accumulation (curves represent total protein accumulated in the culture system, bars represent amount of protein accumulation in each hour. A:iciA, B:hda, C:seqA, D:cspD )

There is no large different between repressive gene transformed group and control group, which indicate that this genes (besides cspD which seems to have some effect) do not have Significant effect on cell metabolism.

Discussion

Four proteins(iciA, hda, seqA, cspD) have good effect in stop bacteria DNA replication initiation, thus overexpress one of this them can slow down the cell cycle. In spite of this, this protein has no significant effect on protein accumulation and max population density.

Future Work

Design experiment to measure more parameter of metabolism. Compare the effect of cycle repressive gene on expression of genes both in chromosome and plasmid.

Reference

[1] Initiation of DNA replication in Escherichia coli. W Messer. J. Bacteriol. 1987, 169(8):3395.