Team:NYMU-Taipei/chassis

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Revision as of 22:00, 28 October 2011 (view source) BarryChen (Talk | contribs) (→Negatively Regulated) ← Older edit		Revision as of 22:02, 28 October 2011 (view source) BarryChen (Talk | contribs) (→MSGM's ingredients) Newer edit →
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	<font size=3>Like ''E. coli'', AMB-1 also grows in liquid medium, and this medium is called '''Magnetic Spirillum Growth Medium (MSGM)'''.  In the medium, we added nutrient, iron source (supplying AMB-1 in magnetite formation within magnetosomes), vitamin, and mineral solution.</font>		<font size=3>Like ''E. coli'', AMB-1 also grows in liquid medium, and this medium is called '''Magnetic Spirillum Growth Medium (MSGM)'''.  In the medium, we added nutrient, iron source (supplying AMB-1 in magnetite formation within magnetosomes), vitamin, and mineral solution.</font>
-	=='''<font size=4><font color=green>MSGM's  ingredients'''</font>==	+	=='''<font size=4><font color=green>(1) MSGM's  ingredients'''</font>==
	<font size=3><font color=red>(In grams per liter)</font></font>		<font size=3><font color=red>(In grams per liter)</font></font>

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Revision as of 22:02, 28 October 2011

• Home Get to HOME
• Idea Our Idea
  Concept Brainstorming Background Review
• Design/ Experiment
  Optomagnetic Design Protein Modelling NeuroSymbiosis Chassis SynthoPrime Lab Protocol
• Results
  Optomagnetic Bacteria NeuroSymbiosis Chassis Parts International Collaboration
• Human Practice & Safety
  Synthetic Neurobiology Human Practice Evaluation & Safety Assessment
• About Us
  The School The Team China Meetup Acknowledgement

Plasmid Backbone We name it pYMB...

(1) Background and Origin

In order to create a shuttle vector for manipulating and transforming gene into Magnetospirillum magneticum AMB-1, we found sound theoretical supports from papers published from Matsunaga T.’s laboratory in Tokyo University of Agriculture and Technology. Under their research and information support, we know that the construct of pUMG is very simple, with pUC19 ligated on BamHI site with pMGT, an endogenous plasmid found in Magnetospirillum magneticum MGT-1.Therefore, the construction of a new shuttle vector named pYMB is our primary aim of our cooperative initiative.

(2) Structure and Assembly

pYMB (Fig. 1) is described to contain the ori (origin of replication) and rep gene (required for replication) of pMGT. Once the synthetic work has been done, pYMB is constructed by equipping the ori, the appropriate promoter for AMB-1 (Pmms16 and Pmsp3 as our candidate) and rep gene on the commercial plasmid pUG19 which was revised as the expression of Biobrick backbone psB1A1with promotor Pmsp1. The constructed vector is capable of replicating within both E. coli and AMB-1, fully sufficing a competent shuttle vector for genetic engineering the magnetotactic bateria.

Fig.1 pYMB Work Flow
The way we design the shuttle vector for magnetic bacteria

(3) Related Parts

●Succinic acid             0.74 g
●KH2PO4                    0.68 g
●Sodium nitrate            0.12 g
●Sodium thioglycolate      0.1 g
●*Wolfe's vitamin solution  10 ml(stock)
●*Wolfe’s mineral solution  5  ml(stock)
●Ferric quinate solution   2  ml(0.27 g of FeCl3 and 0.19 g of quinic acid in 100 ml water)
 Adjust pH to 7.0 with NaOH

●Succinic acid             0.74 g
●KH2PO4                    0.68 g
●Sodium nitrate            0.12 g
●*Wolfe's vitamin solution  10 ml(stock)
●*Wolfe’s mineral solution  5  ml(stock)
●Ferric quinate solution   2  ml(0.27 g of FeCl3 and 0.19 g of quinic acid in 100 ml water)
●L-cysteine                0.005%(Instead of sodium thioglycolate) 
●Yeast extract             0.01%
●Polypeptone               0.02%
 Adjust pH to 7.0 with NaOH

● Step1: Harvested and washed AMB-1 with 10 mM TES buffer containing 272 mM sucrose (pH 7.5).

● Step2: Resuspended AMB-1 in the same buffer at 10^9 cells/ml.

● Step3: Transferred to 500 micro-liter of MSGM supplemented with 20 mM Mg2+ and 
incubated at 27°C overnight with shaking at 100 rpm.

● Step4: Diluted in 5 ml of MSGM containing 0.7% agar.

● Step5: Plated on 1% agar in MSGM (supplied with ampicillin at 5 ug/ml or kanamycin at 2.5 ug/ml) 
incubated under anaerobic conditions.