Part:BBa_K2120310

B0015+CI+B0034+pBAD-araC+B0015+pR+B0032+RFP+B0015

pBAD is a promoter induced by arabinose and araC is the repressor. We added different concentration of arabinose to induce the pBAD promoter to express different concentration of inhibitor. And cI is an inhibitor which is built to repress the expression of pR promoter. We use it to control the RFP intensity expressed by pR promoter. RFP is the reporter gene. We change the promoter direction and add another B0015 to optimize the circuits. We reserve BBa_K2120305 to avoid it affect the expression of RFP. Compared with BBa_K2120309, We add B0015 between araC and pR promoter.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1001
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1166
  Illegal AgeI site found at 2925
  Illegal AgeI site found at 3037
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1183
  

P-Slackiller is the iGEM 2016 project of BIT-China. In industrial produce, plasmid plays an important role in produce because it carries function gene which codes something we wanted. But bacteria will lose the functional plasmid during the division then become a “Slacker” decreasing the efficiency. In order to kill the “Slacker”, we aim to design logical gene switch to sense the copy number variety.

However, it is difficult to control the plasmid copy number.In order to know the relationship between the inhibitor and in-promoter, we use the arabinose induced promoter BBa_K808000 to express the inhibitor.So we can add arabinose with different concentrations to induce the promoter and create an environment with different concentrations of intracellular inhibitor to find the relationship between inhibior and in-promoter.

The logical switch we select the relationship of inhibitor-promoter. In our building work, we finally decide two kinds of inhibitor-promoter, cI-pR and tetR-pTet. Then we add RFP in the downstream of promoter as a reporter for indicating “on” or “off”.

First, we built K2120309. But during our test, we found the expression of araC would influence the expression of RFP. So we added another B0015 between araC and pR.

Beside, we also have built a similar device, BBa_K2120311, using tetR-pTet. But we had no time to test it.

Usage and biology

Inducer: L(+)-arabinose

There is a liner relationship between inducer and expression of cI. But it is not a strict liner relationship (please visit BBa_K2120302 page).

With increasing the arabinose concentration from 0 to 0.005%, the tansciption level of pR will decrease. And we can estimate the relationship between inhibitor (cI) and promoter for adjusting the switch’s “on” and “off”.

Test

We transformed the plasmids, pSB1K3, contained this device into E.coli TOP 10. And we tested it in LB medium with different arabinose concentration. Firstly, the two strains containing empty pSB1K3 and containing the device on pSB1K3 separately overnight (12 h) growth in the LB medium. Then the overnight culture was diluted 1:100 into new LB medium while add the certain concentration of the arabinose

And we measured the fluorescence intensity and OD600 at the same time. Then we get the single cell’s fluorescence intensity by fluorescence intensity being divided by OD600.

Result

We repeated the test twice. The same as result of K2120302, at the beginning of the growth, cells do not show different fluorescence strength until 6-7 hours. We found the OD600 is preserved at 2.0-2.5 when cell grew for 4-5 hours after induced.

And at different arabinose concentration, group A and group B both show that expression of RFP does not correspond to inducer until after induced 15 -16 hours. (Fig 1 and Fig 2)

We draw a fluorescence-arabinose concentration curve, Fig 3. The twice test have a similar tendency when induced different arabinose. And the curve displays that the expression of RFP stops decreasing at 0.003% arabinose. So we can get a conclusion, the transcription of pR being repressed when the expression level of cI induced by corresponding arabinose concentration. In other words, the logical switch (cI-pR) will be turned “off” when arabinose concentration is higher than 0.003%. On the contrary, when the arabinose concentration is lower than 0.003%, this switch is turned “on”.

Combine with the result of K2120302, we can estimate the relationship between inhibitor and promoter for further “P-Slackiller” design and experiment.