Team:Groningen/Notebook/30 August 2009

From 2009.igem.org

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Weekend stuff

Metal accumulation

ON cultures
Growth Name Plasmid Inoculation date Antibiotics
NO
pBAD pSB1AC3? 29august2009 LB-Cam
YES
pBAD pSB1AC3? 29august2009 LB-Amp
NO
pLac pSB1AC3? 29august2009 LB-Cam
YES
pLac pSB1AC3? 29august2009 LB-Amp
YES
fMT #6 pSB1AC3 29august2009 LB-Amp
YES
SmtA #3 pSB1AC3 29august2009 LB-Amp
YES
pLow-fMT #3 pSB1AC3 29august2009 LB-Amp
YES
pLow-fMT #4 pSB1AC3 29august2009 LB-Amp
YES
pLac-fMT #2 pSB1AC3 29august2009 LB-Amp

pSB1AC3 There was doubt on the identity of the vector in which the inducible promotors were build, seeing as it did not grow on LB supplemented with chloramphenicol it is most probably not pSB1AC3, perhaps pSB1A2. Cultures were put in glycerol stocks, except for pBAD & pLac whose identity first need to be confirmed.

Glycerol stocks (-80 °C)

Component Description Part or Accession # Base Pairs (bp) Plasmid (backbone) Resistance Well Quality control Storage Medium+Antibiotics Organism Date of storage
pLac-fMT Metallothionein with inducible promotor Part or Accession # Base Pairs (bp) pSB1AC3 Ampicillin/chloramphenicol plate 3, pos. 55 Quality control LB-Ampicillin E. coli TOP10 30august2009
pLow-fMT #4 Metallothionein with low constitutive promotor Part or Accession # Base Pairs (bp) pSB1AC3 Ampicillin/chloramphenicol plate 3, pos. 56 Quality control LB-Ampicillin E. coli TOP10 30august2009
pLow-fMT #3 Metallothionein with low constitutive promotor Part or Accession # Base Pairs (bp) pSB1AC3 Ampicillin/chloramphenicol plate 3, pos. 57 Quality control LB-Ampicillin E. coli TOP10 30august2009
SmtA Metallothionein Part or Accession # Base Pairs (bp) pSB1AC3 Ampicillin/chloramphenicol plate 3, pos. 58 Quality control LB-Ampicillin E. coli TOP10 30august2009
fMT #6 Metallothionein Part or Accession # Base Pairs (bp) pSB1AC3 Ampicillin/chloramphenicol plate 3, pos. 59 Quality control LB-Ampicillin E. coli TOP10 30august2009

Transformation

SmtA+promotor
Colonies Name Plasmid Date of plating Antibiotics
0
Negative control (50 μL) 1 μL MilliQ 29august2009 LB-Amp100
0
Negative control (200 μL) 1 μL MilliQ 29august2009 LB-Amp100
0
Positive control (50 μL) 1 μL pSB1AC3+high constitutive promotor 29august2009 LB-Amp100
0
Positive control (200 μL) 1 μL pSB1AC3+high constitutive promotor 29august2009 LB-Amp100
4
SmtA+pLac (50 μL) pSB1AC3 29august2009 LB-Amp100
22
SmtA+pLac (200 μL) pSB1AC3 29august2009 LB-Amp100
37
SmtA+pLow (50 μL) pSB1AC3 29august2009 LB-Amp100
~72
SmtA+pLow (200 μL) pSB1AC3 29august2009 LB-Amp100

pSB1AC3 was in fact pSB3K3! Therefore no colonies could grow.
5 colonies of each promotor were inoculated for an ON culture, colony PCR was postponed to monday.

Transporters

HmtA

the second PCR1.2 and PCR2 worked


April
MTWTFSS
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30