Team:USTC/Results

From 2008.igem.org

Correlation between GFP expression and cell density

In order to know how the expression amount of signal molecules is related to cell density, we use GFP as the intermediate variable between the two variables. First, we determined the function of GFP according to the population of bacteria. Here, we have constructed the part with BioBricks R0040 and E0840. After ligating them together, we measured the amount of GFP expression and corresponding OD values and then worked out the correlation curve using Origin 7.5.

Fig1: OD600 increases exponently as to time.

Fig2: The relation between GFP expression and OD600.

• From the figures above, we can draw the conclusion that the expression of GFP generally takes on a linear function of OD when the OD600 is between 0.2 to 0.9.

Function Test

• After successfully cloning GFP(LVA), we try to test its function.We ligate it with lac promoter R0010,a strong RBS B0034 and teminator B0015. We transform the plasmid with these parts to E.coli strain TOP10 and induce the GFP expression with 0.1mM IPTG.

fig 3.Cells grow on the left plate generate GFP after IPTG induced,celles on the right plate is negtive control.

fig 8. Cells that generate GFP in fluorescence microscope

• Because LacI is recruited here to regulate our system, IPTG should be absent. As it is already known that there is LacI genes in the genome of E.coli strain TOP10, we performed some experiments to test whether the influence can be big enough to affect our results.

fig 4.This figure shows that though lacI gene exists in E.coli genome, GFP do expresses without IPTG because of the high copies of the plasmids.

fig 5.Cells express GFP with IPTG induced.

fig 10.Though much weaker,Cells do express GFP under control of Lac promoter without IPTG.

• We then ligate this unit with the unit R0062-B0034-K082005-B0015 which gives weak constangly expression of a weak LacI mutant LacIM1.We use different concentration of IPTG to induce the expression of GFP.Result show as the fig 6

fig 6

• We have constructed some sequence with the purpose of testing whether the RNA polymerase can transcribe the target gene. For instance, we have built the following parts:

1. R0040-E0840;
2. R0040-B0034-C0070-E0840;
3. R0040-B0034-K082004-E0840;
4. R0071-E0840.

As expected, the plasmid containing the first three sequences can generate green fluerescence while the view field remains dark when the bacteria contain the fourth sequence.

• The recombinase system is successfully constructed. The two pictures below show the results.The left one is Cre+GFP ,and the right one is lox71+GFP+lox66. They both have good performances.Co-transformation will be done next.

Fig 7.Co-expression with Cre and GFP.(taken by Lu Xie)

Fig 8.Expression GFP on the lox71+lox66 system.(taken by Lu Xie)

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