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 use it to test the expression of reporter gene in our validation circuit .And CI is an inhibitor which is built to repress the expression of PR promoter. We use it to control the “on” or ”off” state of toxin gene.RFP is the reporter gene. In order to test these do not interfere with each other , We reserve BBa_K2120305. Compared with BBa_K2120309,We add B0015 between AraC and pR.

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. So we choose BBa_K808000 whose expression level of downstream gene can vary continuously for imitating the change of plasmid copy numbers. And connect the logical switch downstream.

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. We add 2ml activated bacteria into new LB medium and induce by adding arabinose. The negative control is pSBIK3 without K2120310. The positive control is the bacteria contained K2120310 but no arabinose.

And we measured the fluorescence intensity and OD600 at the same time. The fluorescence measurement is carried by Biotek plate reader. Then we get the single cells’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.