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Team:Groningen/Notebook/3 September 2009
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''' EM pictures ''' | ''' EM pictures ''' | ||
| - | :→ {{ | + | :→ {{done}} Today we will try to get some Electon microscopy pictures of the gas vesicle producing bacteria from the ON cultures. |
Orinigal pNL29 iptg-GVP and the biobricks ars-GVP and | Orinigal pNL29 iptg-GVP and the biobricks ars-GVP and | ||
| Line 17: | Line 17: | ||
:→ {{todo}} Test control of bouyancy in Saline solution (grow plates with GVP constructs) | :→ {{todo}} Test control of bouyancy in Saline solution (grow plates with GVP constructs) | ||
| - | :→ {{ | + | :→ {{done}} Order synthetic DNA for GVP |
:→ {{todo}} Order primer for PstI site removal | :→ {{todo}} Order primer for PstI site removal | ||
| - | :→ {{ | + | :→ {{done}} Test promoter strenght compared to BBa_J23101 promoter (Sven) |
:→ {{todo}} Enter sequences of constructs to Sandbox | :→ {{todo}} Enter sequences of constructs to Sandbox | ||
| Line 188: | Line 188: | ||
'''Restriction for Assembly''' | '''Restriction for Assembly''' | ||
| - | The | + | The vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61035 BBa_J61035] containing the [http://partsregistry.org/Part:BBa_K190015 pArsR], [http://partsregistry.org/Part:BBa_K190016 pZntR], and [http://partsregistry.org/Part:BBa_K190017 pCueO] with [http://partsregistry.org/Part:BBa_I750016 GVP] composite parts were cut with PstI and EcoRI to create correct ends for insert into [http://partsregistry.org/Part:pSB2K3 pSB2K3], which was also cut with EcoRI and PstI (4x). |
{|cellpadding="2" cellspacing="1" border="4" | {|cellpadding="2" cellspacing="1" border="4" | ||
| Line 265: | Line 265: | ||
Restriction was kept at 37C for 40 min. and put on ice until used for gel purification. | Restriction was kept at 37C for 40 min. and put on ice until used for gel purification. | ||
| + | |||
| + | |||
| + | [[Image:Zymoclean Gel DNA Recovery Kit (D4001) 2.jpg|thumb|300px| www.zymoresearch.com]] | ||
'''Purification''' | '''Purification''' | ||
| - | [[Image:3-9 no.1.jpg| | + | [[Image:3-9 no.1.jpg|450px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]] |
:→ From left to right: 1kB ladder, pSB2K3 (no.1, no.2, no.3, and no.4), J61035-pArsR-GVP , Empty Slot, J61035-pZntR-GVP, Empty Slot, J61035-pCueO-GVP | :→ From left to right: 1kB ladder, pSB2K3 (no.1, no.2, no.3, and no.4), J61035-pArsR-GVP , Empty Slot, J61035-pZntR-GVP, Empty Slot, J61035-pCueO-GVP | ||
| + | :→ The red lines indicate the expected position of the pSB2K3 fragments, ~4400bp for vector and ~1070bp for RFP fragment. The size of the RFP fragment seems to be correct, but the vector part is ~1500bp to small. It can be caused by the slight overloading of the gel, but for the ligation products of tomorrow extra care must be taken to be sure of the vector. | ||
| - | + | :→ One thing that suggest it must be the vector is its resistance for kanamycin, and the high amount of plasmid after growth with IPTG. | |
| - | + | ||
| + | A "Zymoclean(TM) Gel DNA Recovery Kit" standard protocol was used. | ||
| + | |||
| + | * In step 7 an amount of 10μL MQ was added to elute the DNA fragments. | ||
| Line 288: | Line 295: | ||
|'''Restriction Control''' | |'''Restriction Control''' | ||
|- | |- | ||
| - | | | + | |pSB2K3 (7C) no.1 (EcoRI,PstI) |
| - | | | + | |37.7 |
| - | | | + | |1.84 |
| - | | | + | |0.52 |
| - | | | + | |fridge |
| - | | | + | |See Gel |
| + | |- | ||
| + | |pSB2K3 (7C) no.2 (EcoRI,PstI) | ||
| + | |42.9 | ||
| + | |1.87 | ||
| + | |1.90 | ||
| + | |fridge | ||
| + | |See Gel | ||
| + | |- | ||
| + | |pSB2K3 (7C) no.3 (EcoRI,PstI) | ||
| + | |32.9 | ||
| + | |1.84 | ||
| + | |1.52 | ||
| + | |fridge | ||
| + | |See Gel | ||
| + | |- | ||
| + | |pSB2K3 (7C) no.4 (EcoRI,PstI) | ||
| + | |51.2 | ||
| + | |2.07 | ||
| + | |0.07 | ||
| + | |fridge | ||
| + | |See Gel | ||
| + | |- | ||
| + | |pArsR-GVP (EcoRI,PstI) | ||
| + | |8.9 | ||
| + | |2.06 | ||
| + | |0.03 | ||
| + | |fridge | ||
| + | |See Gel | ||
| + | |- | ||
| + | |pZntR-GVP (EcoRI,PstI) | ||
| + | |22.6 | ||
| + | |1.71 | ||
| + | |1.01 | ||
| + | |fridge | ||
| + | |See Gel | ||
| + | |- | ||
| + | |pCueO-GVP (EcoRI,PstI) | ||
| + | |17.3 | ||
| + | |1.83 | ||
| + | |0.55 | ||
| + | |fridge | ||
| + | |See Gel | ||
|} | |} | ||
| Line 303: | Line 352: | ||
*Send pLac-fMT for sequencing | *Send pLac-fMT for sequencing | ||
===Vectors=== | ===Vectors=== | ||
| + | |||
| + | ===TEM=== | ||
| + | ''' Transmission electron microscopy ''' | ||
| + | |||
| + | The three pictures below show pNL29 induced with iptg for one day. (1) magnification 22.000X (2) magnification 35.000X (3) magnification 260.000X on one vesicle. | ||
| + | |||
| + | <gallery> | ||
| + | Image:Gas-ves-004.tif.png|1 | ||
| + | Image:Gas-ves-002.tif.png|2 | ||
| + | Image:Gas-ves-005.tif.png|3 | ||
| + | </gallery> | ||
| + | |||
| + | In the first two picture we see some light round structures comparable to the structures shown in the Li and Cannon paper. The third picture is what we believe can be a gas vesicles (outside of the cell). | ||
| + | Unfortunately these cells have a cellwall. Next week we will make pictures of a protoplast, we hope to reproduce the papers result more clearly. | ||
==Dry== | ==Dry== | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Latest revision as of 09:15, 7 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
LB-medium is badly needed!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
GVP Cluster
EM pictures
- → DONE Today we will try to get some Electon microscopy pictures of the gas vesicle producing bacteria from the ON cultures.
Orinigal pNL29 iptg-GVP and the biobricks ars-GVP and
- → TODO Choose colonies from plates for growth of E.coli TOP10 with GVP behind LAC/pBAD (+RBS) promoter in pSB1AC3
- → TODO Make glycerol stocks of pBad/araC, pBad/araC+RBS+GVP, and pLacI+RBS+GVP (both in pSB1A2 and pSB1AC3 plasmid)
- → TODO Test control of bouyancy in Saline solution (grow plates with GVP constructs)
- → DONE Order synthetic DNA for GVP
- → TODO Order primer for PstI site removal
- → DONE Test promoter strenght compared to BBa_J23101 promoter (Sven)
- → TODO Enter sequences of constructs to Sandbox
Colonies on Plates
| Name | Plasmid Used | Antibiotics on Plasmid | No. of Colonies | Date |
| pLacI-GVP | http://partsregistry.org/Part:pSB1A2 pSB1A2] | Ampicillin | 26 | 3/9 |
| pLacI-GVP (concentrated) | http://partsregistry.org/Part:pSB1A2 pSB1A2] | Ampicillin | ~100 | 3/9 |
| pBad/araC-GVP | http://partsregistry.org/Part:pSB1A2 pSB1A2] | Ampicillin | 1 | 3/9 |
| pBad/araC-GVP (concentrated) | http://partsregistry.org/Part:pSB1A2 pSB1A2] | Ampicillin | ~40 | 3/9 |
| Negative Control | MQ | None | 0 | 3/9 |
| Positive Control | J61002-J23101 | Ampicillin | ~2000 | 3/9 |
- → The plates showed normal colony growth.
O.n. precultures
- → All twelve o.n. precultures showed bacterial growth, and could be used to isolate plasmid. The first four are registry vector pSB2K3 with RFP as reporter (plate 1, 7C) which were transformed on tuesday. The isolated plasmid is used to house all GVP constructs to combine with the vector pSB1AC3 with transporter and accumulation genes.
- → The next eight tubes contain our own designed biobricks and isolated plasmid will be sent to the registry on friday!
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids 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 4mL 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 30μL MQ and stored in the fridge
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pSB2K3 (7C) no.1 | 178.1 | 1.87 | 2.32 | D-1 | Yes (EcoRI/PstI) |
| pSB2K3 (7C) no.2 | 206.8 | 1.89 | 2.34 | D-2 | Yes (EcoRI/PstI) |
| pSB2K3 (7C) no.3 | 147.1 | 1.87 | 2.31 | D-3 | Yes (EcoRI/PstI) |
| pSB2K3 (7C) no.4 | 186.2 | 1.85 | 2.15 | D-4 | Yes (EcoRI/PstI) |
| BBa_K190015 | 155.3 | 1.88 | 2.14 | B-1 | Yes (EcoRI/PstI) |
| BBa_K190016 | 69.4 | 1.85 | 2.28 | B-2 | Yes (EcoRI/PstI) |
| BBa_K190017 | 85.8 | 1.90 | 2.22 | B-3 | Yes (EcoRI/PstI) |
| BBa_K190022 | 94.9 | 1.82 | 1.70 | B-4 | Yes (EcoRI/PstI) |
| BBa_K190023 | 90.4 | 1.87 | 2.03 | B-5 | Yes (EcoRI/PstI) |
| BBa_K190024 | 82.2 | 1.84 | 2.06 | B-6 | Yes (EcoRI/PstI) |
| BBa_K190025 | 297.1 | 1.85 | 2.37 | B-7 | Yes (EcoRI/PstI) |
| BBa_K190026 | 13.2 | 1.91 | 2.20 | B-8 | Yes (EcoRI/PstI) |
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61035 BBa_J61035] containing the [http://partsregistry.org/Part:BBa_K190015 pArsR], [http://partsregistry.org/Part:BBa_K190016 pZntR], and [http://partsregistry.org/Part:BBa_K190017 pCueO] with [http://partsregistry.org/Part:BBa_I750016 GVP] composite parts were cut with PstI and EcoRI to create correct ends for insert into [http://partsregistry.org/Part:pSB2K3 pSB2K3], which was also cut with EcoRI and PstI (4x).
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pSB2K3 (7C) no.1 | 6.0 | 10.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 (7C) no.2 | 6.0 | 10.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 (7C) no.3 | 6.0 | 10.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 (7C) no.4 | 6.0 | 10.0 | 3.0 | 1.0 | x | x | 1.0 |
| J61035-pArsR-GVP | 16.0 | x | 3.0 | 1.0 | x | x | 1.0 |
| J61035-pZntR-GVP | 10.0 | 6.0 | 3.0 | 1.0 | x | x | 1.0 |
| J61035-pCueO-GVP | 10.0 | 6.0 | 3.0 | 1.0 | x | x | 1.0 |
Restriction was kept at 37C for 40 min. and put on ice until used for gel purification.
_2.jpg)
Purification
- → From left to right: 1kB ladder, pSB2K3 (no.1, no.2, no.3, and no.4), J61035-pArsR-GVP , Empty Slot, J61035-pZntR-GVP, Empty Slot, J61035-pCueO-GVP
- → The red lines indicate the expected position of the pSB2K3 fragments, ~4400bp for vector and ~1070bp for RFP fragment. The size of the RFP fragment seems to be correct, but the vector part is ~1500bp to small. It can be caused by the slight overloading of the gel, but for the ligation products of tomorrow extra care must be taken to be sure of the vector.
- → One thing that suggest it must be the vector is its resistance for kanamycin, and the high amount of plasmid after growth with IPTG.
A "Zymoclean(TM) Gel DNA Recovery Kit" standard protocol was used.
- In step 7 an amount of 10μL MQ was added to elute the DNA fragments.
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pSB2K3 (7C) no.1 (EcoRI,PstI) | 37.7 | 1.84 | 0.52 | fridge | See Gel |
| pSB2K3 (7C) no.2 (EcoRI,PstI) | 42.9 | 1.87 | 1.90 | fridge | See Gel |
| pSB2K3 (7C) no.3 (EcoRI,PstI) | 32.9 | 1.84 | 1.52 | fridge | See Gel |
| pSB2K3 (7C) no.4 (EcoRI,PstI) | 51.2 | 2.07 | 0.07 | fridge | See Gel |
| pArsR-GVP (EcoRI,PstI) | 8.9 | 2.06 | 0.03 | fridge | See Gel |
| pZntR-GVP (EcoRI,PstI) | 22.6 | 1.71 | 1.01 | fridge | See Gel |
| pCueO-GVP (EcoRI,PstI) | 17.3 | 1.83 | 0.55 | fridge | See Gel |
Transporters
Metal Accumulation
- Send MBP-ArsR for sequencing again
- Send pLow-fMT for sequencing
- Send pLac-fMT for sequencing
Vectors
TEM
Transmission electron microscopy
The three pictures below show pNL29 induced with iptg for one day. (1) magnification 22.000X (2) magnification 35.000X (3) magnification 260.000X on one vesicle.
 1 |
 2 |
 3 |
In the first two picture we see some light round structures comparable to the structures shown in the Li and Cannon paper. The third picture is what we believe can be a gas vesicles (outside of the cell). Unfortunately these cells have a cellwall. Next week we will make pictures of a protoplast, we hope to reproduce the papers result more clearly.
Dry
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