Team:Groningen/Notebook/14 August 2009

From 2009.igem.org

(Difference between revisions)
m (New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== :→ {{todo}} make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultu...)
(→Dry)
 
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===GVP Cluster===
===GVP Cluster===
-
:→ {{todo}} make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown)
+
:→ {{done}} make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown)
-
:→ {{todo}} isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures
+
:→ {{done}} isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures
-
:→ {{todo}} make a planning for next week
+
:→ {{done}} make a planning for next week
 +
 
 +
'''Plates'''
 +
 
 +
Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth.
 +
 
 +
:→ The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at 37°C for an additional 4 hours to be sure. Still no growth after addition incubation at 37°C, can be due to high energy cost of vesicle production resulting in the death of the cells!
 +
 
 +
'''Over Night Cultures'''
 +
 
 +
:→ All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. From each culture 800uL was added to 300uL 87% glycerol and frozen in liquid nitrogen. Stocks were stored at position 21 to 24 in the -80°C fridge.
 +
 
 +
[[Image:Plasmid Miniprep Kit Flow Protocol.gif|thumb|150px| www.sigmaaldrich.com]]
 +
 
 +
'''Plasmid Purification'''
 +
 
 +
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoters with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
 +
 
 +
* From each tube 8mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
 +
* Plasmids were eluted with 50μL MQ and stored in the fridge
 +
 
 +
'''Concentration'''
 +
 
 +
{|cellpadding="2" cellspacing="1" border="4"
 +
|'''Plasmid'''
 +
|'''Conc. ng/μL'''
 +
|'''260/280 
 +
|'''260/230  '''
 +
|'''-20 box (michael
 +
|'''Restriction Control'''
 +
|-
 +
|BBa_J61002-BBa_J23100 (high+RFP)
 +
|288.1
 +
|1.90
 +
|2.33
 +
|D-8
 +
|No
 +
|-
 +
|BBa_J61002-BBa_J23106 (med.+RFP)
 +
|162.2
 +
|1.93
 +
|2.31
 +
|D-9
 +
|No
 +
|-
 +
|BBa_J61002-BBa_J23109 (low+RFP)
 +
|114.2
 +
|1.93
 +
|2.28
 +
|D-10
 +
|No
 +
|}
===Transporters===
===Transporters===
-
===Metal Accumulation===
+
[[Image:F102471_2009-08-14_12hr_03min_PCR1_PCR2_Dynazyme_XbaI.jpg|thumb|PCR2, PCR1 Dynazyme XbaI]]
 +
====HmtA====
 +
After 2 weeks of PCR-ing plasmid isolations there is a product worth wile to send for sequencing. 4,5,6, and 8
 +
In the first 4 lanes  PCR2 (261bp) is created from HmtA-AC3 plasmid with PstI 's left in place. In lane 5 till 8 PCR1 (1153bp)is made from HmtA-AC3 plasmid with PstI 's left in place to make mega primers to mutate the restriction sites.
 +
 +
In the lower left quarter is a PCR on HmtA-AC3 plasmid with PstI 's left in place with VF2 VR and failed. 
 +
The second lower quarter is a XbaI restriction control on plasmids 4,5,6 and 8 not completely digested but indicating the construct is correct in the way that there is a restriction site of the bio brick, meaning the EcoRI mutation got removed.
 +
 +
===Metal Accumulation===
 +
*'''ligate MymT in pSB1AC3'''
 +
**Purify mymT-RBS-pre/suff from gel using Roche Gel Extraction kit
 +
**Elute in 25uL TE buffer (pH8)
 +
**Digest mymT-RBS-pre/suff with EcoRI and Spe I
 +
**Ligate with pSB1AC3/EcoRI-SpeI
===Vectors===
===Vectors===
==Dry==
==Dry==
 +
Jasper changed [[Team:Groningen/Modelling/Arsenic|the model]] to take dimeric binding of ArsR into account and to ignore ArsD, as well as putting an image map on-line as the first step towards a clearer interface. The new model leads to a number of third order equations, which means that it is no longer feasible to give closed-form solutions, but it does seem perfectly feasible to compute the solutions numerically (as demonstrated by the updated calculator on the [[Team:Groningen/Project/Accumulation|accumulation page]]).
{{Team:Groningen/Notebook/Day/Footer}}
{{Team:Groningen/Notebook/Day/Footer}}

Latest revision as of 06:49, 17 August 2009

Igemhomelogo.png

April
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
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

Wet

GVP Cluster

→ DONE make glycerol stocks for pSB1AC3-BBa_J23106-BBa_I750016 and pSB1AC3-BBa_J23109-BBa_I750016 (two o.n. cultures of each are grown)
→ DONE isolate plasmid J61002-BBa_J23100, J61002-BBa_J23106, and J61002-BBa_J23109 from o.n. cultures
→ DONE make a planning for next week

Plates

Showed single colony growth on plates with pSB3K3-H/L-GVP plasmids, and were stored in the fridge for future preculture growth.

→ The plates with pSB1AC3-H-GVP plasmid showed no growth, and were stored at 37°C for an additional 4 hours to be sure. Still no growth after addition incubation at 37°C, can be due to high energy cost of vesicle production resulting in the death of the cells!

Over Night Cultures

→ All cultures showed growth, and can be used for plasmid isolation and glycerol stocks. From each culture 800uL was added to 300uL 87% glycerol and frozen in liquid nitrogen. Stocks were stored at position 21 to 24 in the -80°C fridge.
www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids BBa_J61002 with high, medium and low constitutive promoters with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 8mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
  • Plasmids were eluted with 50μL MQ and stored in the fridge

Concentration

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
BBa_J61002-BBa_J23100 (high+RFP) 288.1 1.90 2.33 D-8 No
BBa_J61002-BBa_J23106 (med.+RFP) 162.2 1.93 2.31 D-9 No
BBa_J61002-BBa_J23109 (low+RFP) 114.2 1.93 2.28 D-10 No

Transporters

PCR2, PCR1 Dynazyme XbaI

HmtA

After 2 weeks of PCR-ing plasmid isolations there is a product worth wile to send for sequencing. 4,5,6, and 8

In the first 4 lanes PCR2 (261bp) is created from HmtA-AC3 plasmid with PstI 's left in place. In lane 5 till 8 PCR1 (1153bp)is made from HmtA-AC3 plasmid with PstI 's left in place to make mega primers to mutate the restriction sites.

In the lower left quarter is a PCR on HmtA-AC3 plasmid with PstI 's left in place with VF2 VR and failed. The second lower quarter is a XbaI restriction control on plasmids 4,5,6 and 8 not completely digested but indicating the construct is correct in the way that there is a restriction site of the bio brick, meaning the EcoRI mutation got removed.

Metal Accumulation

  • ligate MymT in pSB1AC3
    • Purify mymT-RBS-pre/suff from gel using Roche Gel Extraction kit
    • Elute in 25uL TE buffer (pH8)
    • Digest mymT-RBS-pre/suff with EcoRI and Spe I
    • Ligate with pSB1AC3/EcoRI-SpeI

Vectors

Dry

Jasper changed the model to take dimeric binding of ArsR into account and to ignore ArsD, as well as putting an image map on-line as the first step towards a clearer interface. The new model leads to a number of third order equations, which means that it is no longer feasible to give closed-form solutions, but it does seem perfectly feasible to compute the solutions numerically (as demonstrated by the updated calculator on the accumulation page).


April
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
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
Retrieved from "http://2009.igem.org/Team:Groningen/Notebook/14_August_2009"