Part:BBa_K255002

pTet.lacI-cfp(fusion).terminator.pTet.yfp.terminator

It has got single negative feedback loop on the plasmid(pSB2K3) copy number.Plasmid copy numberis dependent on IPTG concentration (inducer). Here there is no control on the lacI expression.The copy number is characterized by YFP while LacI is characterized by LacI-CFP fusion protein. YFP was measured using FACS and the distribution obtained was independent of IPTG. Beta-galactosidase was also measured at various lactose concentration to characterize the expression of gene in the host genome. Further the growth of the transformant was also characterized at different lactose concentrations.

Characterization

Steady state concentration of YFP was measured using FACS. Illustrative FACS results are given below: The mean, variance and standard error were noted for difference strains and at different IPTG concentration. The FACS results are shown for varying IPTG concentrations varying from 0um to 500um.

Result

The YFP Expression increased around 10 folds from 0 to 100 μM of IPTG Conc and thereafter its value was almost constant to 200 and 500 μM of IPTG conc(fig.1).

Figure 1. Expression of BBa_K255002 in lacI deleted E.coli strain

After this the expression profile of YFP between 0 and 100 μM was measured. One can observe that the variability is decreasing as compared to that seen in the open loop(BBa_K255004). Fig.2 shows variation of YFP expression at various IPTG concentrations in Strain BBa_K255002, where the copy number is regulated. YFP indicates the status of copy number

Figure 2.Variation of YFP expression at various IPTG concentrations in Strain BBa_K255002.

As a control, FACS was done with host strain to quantitate the autofluorescence. It was observed that cells are showing autofluorescence but its intensity is very less than our BBa_K255002 strain (fig.3)

Figure 3. Autofluorescence

Safety

Because this part is from commonly used elements in bacteria. No safety problem can be arisen by this part.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1161
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]