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Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter
Analysis of the transformation we did yesterday
Number of colonies and flurorescence
| Ligation name
| Description
| Antibio
| Number Colonies observed
| Fluorescence
| Comments
|
| Ligations
|
| L150
| D105(BV) - D146(BI)
pLas - rbs-TetR-GFP Tripart
| Amp
| 3
| No
| Problem
|
| L151
| D147(FI) - D125(FV)
rbs-LasR - Double terminator
| Kan
| 21
| No
| OK
|
| Controls
|
| C1
| D105(BV)
| Amp
| 150
| NO
| OK
|
| C2
| D125(FV)
| Kan
| 2
| No
| OK
|
| Positive Control
| pUC19
| Amp
| 416 (efficiency 4.10^7)
| No
| OK
|
PCR Screening
Protocol
| Well
| Sample
| Expected size
| Measured size
|
| 1
| 1kb ladder
|
|
|
| 2
| Negative control (pUC19)
| nothing
| nothing
|
| 3
| Positive control (MP101.1)
| 768 pb
| 800pb
|
| 4
| L150.1
| 1992pb
| 400pb
|
| 5
| L150.2
|
| 6
| L150.3
|
| 7
| L151.1
| 1229pb
| 1200pb
|
| 8
| L151.2
|
| 9
| L151.3
|
| 10
| L151.4
|
| 11
| L151.5
|
| 12
| L151.6
|
| 13
| L151.7
|
| 14
| L151.8
|
| 15
| 100pb ladder
|
Conclusion :
L150 doesn't success, measured size match with the promoter size only,
we will check the size of L101 by screening and if it doesn't match with the good size (1827pb)
we will try again the ligation : rbs-TetR (S03879) with GFP tripart (E0840).
L151 is ok we have to put a strong promoter before the part rbs-lasR-double terminator :GREAT !
Minipreps & Stocks
- Cultured in 7,5ml of LB with a thoothpick of a colony.
- Culture O/N at 37°C of :
| Miniprep Name
| Ligation name
| Antibio
| Biobricks
| Description
|
| MP161.1
| L150.1
| Amp
|     
| pLas - rbs-TetR-GFP tripart
|
| MP161.2
| L150.2
|
| MP161.3
| L150.3
|
| MP162.1
| L151.1
| Kan
|
| rbs-LasR - Double terminator
|
| MP162.2
| L151.2
|
| MP162.3
| L151.3
|
Analysis of the other transformation we did yesterday
Evaluation of the number of colonies
| Ligation name
| Number of colonies
| Autoligation control
|
| L 143 (Kan)
| 0
| 0
|
| L 144 (Kan)
| 0
| 0
|
| L 145 (Amp)
| 452
| 430
|
| L 146 (Amp)
| ~ 2000
| 430
|
| L 147 (Amp)
| ~3500
| >5000
|
Remarks:
L143 and L144 did not work once again. Maybe there is a problem with the digestion,
because the primers used present only two nucleotides after the restriction sites. what we should
do is cutting pfliL with xbaI and SpeI. As the vector will autoligate, we should use a phosphatase
to prevent this phenomenon.
For L147 and its control (T4), we do not observe any red fluorescence. It means that the digestion work well, because if it was not digested, the strong promoter J23100
should express mRFP!
We will repeat all the ligation anyway.
Construction of OmpR*+RBS and EnvZ*+RBS: transformation
Evaluation of the number of colonies
| Ligation name
| Number of colonies
| Autoligation control
|
| L 148 (Amp)
| ~10000
| ~2000
|
| L 149 (Amp)
| ~750
| ~2000
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