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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun
From 2014.igem.org
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 1 06/02 - 06/08</a></h6> |
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| + | <ul> | ||
| + | <li><b><i>kivD</i></b></li> | ||
| + | <ul> | ||
| + | <li>This week we aim to transform the construct from the parts distribution.</li> | ||
| + | <ul> | ||
| + | <li>Transformation with electrocompotetent cells</li> | ||
| + | <ul> | ||
| + | <li>K538742: <i>pSB1C3-kivD</i> (parts distribution from 2013, plate 1, 24L)</li> | ||
| + | </ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>kivD</i></li> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Sequencing" target="_blank">Sequencing</a> confirmed identities of all parts, but not whole sequence of the parts was covered</li> | ||
| + | <li>Glycerin stock created: <i>E. coli</i> KRX pSB1C3-<i>kivD</i></li> | ||
| + | </ul> | ||
| + | |||
| + | </ul> | ||
| + | </ul> | ||
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| - | <a style="font-size:24px" href="#" | + | <h6><a style="font-size:24px" href="#"Week 2 06/09 - 06/15</a></h6> |
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 3 06/16 - 06/22</a></h6> |
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| - | <a style="font-size:24px" href="# | + | <h6><a style="font-size:24px" href="#">Week 4 06/23 - 06/29</a></h6> |
</div> | </div> | ||
<div class="content"> | <div class="content"> | ||
Revision as of 10:06, 27 August 2014
 |
June |
 |
- kivD
- This week we aim to transform the construct from the parts distribution.
- Transformation with electrocompotetent cells
- K538742: pSB1C3-kivD (parts distribution from 2013, plate 1, 24L)
- Plasmid isolation of kivD
- Sequencing confirmed identities of all parts, but not whole sequence of the parts was covered
- Glycerin stock created: E. coli KRX pSB1C3-kivD
- Transformation of electrocompotetent E.coli KRX cells:
- K538742: pSB1C3-kivD (parts distribution from 2013, plate 1, 24L)
- K539621: pSB1C3-ilvC (parts distribution from 2013, plate 1, 24F)
- K539626: pSB1C3-ilvD (parts distribution from 2013, plate 1, 22J)
- K539627: pSB1C3-alsS (parts distribution from 2013, plate 1, 24H)
- Resulting colonies were selected to start liquid cultures:
- 5 ml of LB in 20 ml reaction tube
- incubation over night at 37°C and 250 rpm
- Plasmid isolation by ThermoScientific MiniPrep
- Sanger sequencing using VF and VR as sequencing primers
- Identities of all parts were confirmed, but not whole sequence of the parts was covered
- Glycerin stocks were created for all four strains:
- E. coli KRX pSB1C3-alsS
- E. coli KRX pSB1C3-ilvC
- E. coli KRX pSB1C3-ilvD
- E. coli KRX pSB1C3-kivD
- pSB1C3-alsS-ilvC-ilvD-kivD
- PCR amplification of all four coding sequences for a Gibson Assembly
- Elongation time: 90 seconds
- Annealing temperature: 55°C
- Combinations of primer and template combinations are listed in the table below. Sequences for Gibson Assembly are introduced by the primers. Additionally the forward primers contain a RBS (BBa_B0034).
- Products were analyzed by agarose gel electrophoresis
- PCR conditions need to be optimized due to low product quality and missing products
| forward primer | reverse primer | template |
|---|---|---|
| fw_pSB1C3_alsS | rv_ilvC_alsS | pSB1C3-alsS |
| fw_alsS_ilvC | rv_ilvD_ilvC | pSB1C3-ilvC |
| fw_ilvC_ilvD | rv_kivD_ilvD | pSB1C3-ilvD |
| fw_ilvD_kivD | rv_pSB1C3_kivD | pSB1C3-kivD |
| fw_kivD_pSB1C3 | rv_alsS_pSB1C3 | pSB1C3-CFP |
