IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/09/10

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Aleksandra
Testing the counter with the Tet resistance (not integrated on the chromosome)
We have incubated an overnight culture of TURBO cells transformed with 2 types of plasmids : pSB1A2 (high copy number, Amp-resistant) with our construct and pSUlib (low copy number, Kan-resistant) with the pBAD/Integrase. The media was LB+Amp+Kan+Glc. 1% glucose prevents the pBAD promoter from leaking.
We diluted this overnight culture 1/100 in LB+Amp+Kan+Glc and waited for the OD to reach 0.4. From now on, this is the culture we were working with. We plated the negative controls before the integrase expression and then induced the culture with 0.2% arabinose for the integrase to be expressed. The integrase would then excise the terminator sequence between the attC sites and result in tetracyclin resistance casette being expressed: P-RBS-LuxR/attC-term-attC/RBS-TetA(C)-RBS-LuxI → P-RBS-LuxR/attC/RBS-TetA(C)-RBS-LuxI.
Next, samples were taken at 1h intervals and plated on Amp+Glc and Amp+Tet+Glc plates at the dilutions of 1/1,000 and 1/10,000. The 1/10,000 Amp+Glc plates served as controls. Here are the results with 2 different constructs, with the ratio of the number of colonies on the Amp+Tet plate to the number of colonies on the Amp (control) plate at each hour.


Ps/sRBS/LuxR/attC-term-attC/wRBS/TetA(C)/sRBS/LuxI
Image:Pss.jpg


Pm/sRBS/LuxR/attC-term-attC/mRBS/TetA(C)/sRBS/LuxI
Image:Pms1.jpg
Image:Pms2.jpg
The negative controls are really negative, so just as we expected there are no Tet resistant cells before the integrase is expressed. After 1h arabinose induction, there are still no Tet-resistant colonies on our plates. We see some Tet-resistant colonies on all other plates starting with 2h induction. This means that at least 2h of arabinose induction is necessary to make one integration pulse.
We would expect the trend to be upward. It seems so on both graphs during the first 4 hours. However, the results at later hours are rather unexpected. This could be explained by several facts :

  • The plates were put into the incubator at 1h intervals but taken out at the same time, so the "6h" plates were incubated 5h less than the "1h" plates. So this could explain why there seem to be less visible colonies on the later plates. When looked under magnification, a lot of microcolonies are seen there, but it was really difficult to count these.
  • We have a hypothesis on why there are colonies of different sizes on the Amp+Tet plates. In fact, the excision and subsequent TetA(C) expression takes place on a high copy number plasmid. Those cells that have more than 1 excised plasmid express the tetracyclin resistance cassette at a higher rate and might be able to grow faster than those cells that have only 1 active TetA(C) copy. Moreover, the growth rate of daughter cells would also depend on the number of excised plasmids they received after segregation. This could also explain why there is no difference in colony size on the Amp plates : all pSB1A2 plasmids express the Amp resistance gene so neither has growth advantage.
  • We didn't expect as much colonies on the Amp (control) plates and din't make further dilutions. However, there's too much of them to count properly, especially on later plates as the culture became more dense. Next time we should make 10^-5 or even 10^-6 dilutions for the controls.


Some of these factors are difficult to take into account for calculating the excision probability, that's why our final counter is meant to be in a single copy, integrated into the chromosome.


Gel extraction and PCR purification

  • attP
  • I13507/pLux/GFP
  • RBS-GFP-tt (E0240)
  • PmS-MTet-LuxI
  • LuxI
  • pLux/LuxI
  • pLux



PCR to check the chromosomal integration (attB lambda primers)

  • PmS Chr 1-5


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