Difference between revisions of "Team:UST Beijing/Project"

Description

We know that E.coli divides in LB medium at 37C around 20-30min per cycle. In this 20-30 minutes, the bacterium has to duplicate its DNA, 4.5 million base pairs, produce every protein of its body another copy, assemble them into structures, and divide into two almost identical copies of individual bacterium.

On top of this, if we introduce a plasmid of 3kb long, 500 copies of it, into every E.coli individual, we would add some pressure to this replicating process. In this plasmid (pB1A3) , a RFP protein expression cassette (part number BBa_J004450) is inserted. The promoter has a lacI operon but in DH5alpha strain, its basal expression seems not under tight LacI control, probably due to weak expression levels of LacI in DH5alpha, and plasmid high copy numbers.

Here is a picture of a group of RFP-expressing cells cultured in LB:

The obvious phenomena is that there is a dramatic variation of RFP expression among individual cells. Some scholars use stochastic models to describe this phenomena. We would like to analyze the problem differently.

We hypothesize:

1)Every cell division is done unevenly: an "old" cell containing leading_strand_synthesized plasmids; a "young" cell containing lagging_strand_synthesized plasmids. Due to the time difference of DNA synthesis, the leading_strand DNA attracts most of existing transcription factors, thus inititates transcription earlier than the lagging_strand DNA after their seperation into daughter cells;

2)The "old" plasmids can express protein faster than "new" plasmids;

3)Both "old" and "young" cells degrade the protein at similar rate;

4)The expression of RFP is in competition of cell division;

5)If for some reason the cell division stops, RFP continues to accumulate in the cell.

If we manipulate ingredients of the cell culture medium, we could change the doubling time of the bacterium. The longer the doubling time, the more RFP will accumulates. There is a switch point where all cells accumulates the same amount of RFP in proportion to its growth, RFP became constant.

Experiment:

To test the hypothesis, we designed a battery of culture medium:

Here are the results (photos and analysis):

PART1

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At the 5-6 conditions, we observed no visible RFP in all cells. At conditions richer than 5-6, we see increased variation of RFP expression in individual cells; at conditions poorer than 5-6, we see decreased variation of RFP in individual cells

PART2

By different color filter to the measurement of light absorption curve determined using yellow filter.The e. coli expression of red fluorescent protein extraction, using homemade instruments to quantitative determination of red fluorescent protein.