Team:Wageningen UR/Journal/week24

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= Week 24: 8 october - 14 october  =
= Week 24: 8 october - 14 october  =
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'''CCMV'''
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----
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''12 October''
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Transformed IPTG_CCMV_NEG into a BL21 strain. Transformed biobrick RFP in pSB1C3 and the one with IPTG + RBS in kanamycin backbone  into DH5A.<br/><br/>
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''14 October''
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Inoculated 10 mL LB with a colony from each transformation plate.
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<br/><br/>
'''GFP modification'''
'''GFP modification'''
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-> no green fluorescence could be seen  
-> no green fluorescence could be seen  
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''15 October''
 
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* 3rd time growing JM109 containing our biobricks BBa_K883702 (GFP coil with an IPTG induced promoter) and BBa_K883703 (GFP coil + His tag with an IPTG induced promoter) and induce expression with IPTG with new medium
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'''PLRV'''
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-> no GFP production could be seen
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''16 October''
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* plate the JM109 samples containing GFPcoil with an IPTG induced promoter and GFPcoil+his tag with an IPTG induced promoter on agarplates containing IPTG
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-> culture containing BBa_K883702 shows green fluorescence
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* grow colonies containing the biobricks BBa_K883702 BBa_K883703 BBa_K883701 and BBa_K883700
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* grow colonies containing BBa_K883702 and BBa_K883703 in duplo once with IPTG in the medium and once adding IPTG (fresh stock solution) to the culture at OD=0.6 
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* miniprep of the colonies containing BBa_K883702 BBa_K883703 BBa_K883701 and BBa_K883700
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* 2nd try to send the bricks BBa_K883702 BBa_K883703 BBa_K883701 and BBa_K883700 in for sequencing
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-> sequencing revealed that BBa_K883702 and BBa_K883703 had the expected sequence but BBa_K883701 and BBa_K883700 where faulty
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''18 October''
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* transformation of BBa_K883702 into BL21 (producing strain) - plating the transformants on plates containing IPTG
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'''Hepatitis B inside modification'''
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''15 October''
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We started another production process to obtain PLRV VLPs. We considered that production of the CCMV VLP with the negative inside had worked well in the first batch which produced with the well acknowledged production strain BL-21, and not in the later batch from the JM-109 strain. Because our last attempt to produce PLRV VLPs was done in JM-109 cells as well, we figure it may be possible that the production strain was the problem. Therefore, we will try the same procedure again but now in BL-21 ''E. coli''.
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* PCR check of the PCR fragment obtained on 27.August
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[[File:PCR check Hepinsidecoil 11.10.png|500px|center|thumb|<p align="justify">''Figure 1: The purified PCR fragment contains fragments of the correct size as well as a bigger fragment. The nonpurified sample shows only a smere.'</p>]]
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* ligation of the PCR product in pJET
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* transformation with DH5α
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-> there was growth on the negative control plate
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We transformed electrocompetent BL-21 cells with Bba_K883401, which contains a PLRV Coat Protein encoding gene under the IPTG inducible promotor.
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[[https://2012.igem.org/Team:Wageningen_UR/Journal/week23 previous week]]          [[https://2012.igem.org/Team:Wageningen_UR/Journal/week25 next week]]
[[https://2012.igem.org/Team:Wageningen_UR/Journal/week23 previous week]]          [[https://2012.igem.org/Team:Wageningen_UR/Journal/week25 next week]]

Latest revision as of 14:58, 26 October 2012

Week 24: 8 october - 14 october

CCMV

12 October

Transformed IPTG_CCMV_NEG into a BL21 strain. Transformed biobrick RFP in pSB1C3 and the one with IPTG + RBS in kanamycin backbone into DH5A.

14 October

Inoculated 10 mL LB with a colony from each transformation plate.

GFP modification

11 October

  • grow JM109 containing our biobricks BBa_K883702 (GFP coil with an IPTG induced promoter) and BBa_K883703 (GFP coil + His tag with an IPTG induced promoter)and induce expression with IPTG

-> no conclusion could be made due to growth on the negative control

  • send samples of BBa_K883702 BBa_K883703 BBa_K883701 and BBa_K883700 in for sequencing to check if the last transformation from pJET into the iGEM backbones succeded

-> sequencing results where never obtained - the samples did not arrive


12 October

  • 2nd time growing JM109 containing our biobricks BBa_K883702 (GFP coil with an IPTG induced promoter) and BBa_K883703 (GFP coil + His tag with an IPTG induced promoter) and induce expression with IPTG

-> again there is growth on the negative control -> no green fluorescence could be seen


PLRV

We started another production process to obtain PLRV VLPs. We considered that production of the CCMV VLP with the negative inside had worked well in the first batch which produced with the well acknowledged production strain BL-21, and not in the later batch from the JM-109 strain. Because our last attempt to produce PLRV VLPs was done in JM-109 cells as well, we figure it may be possible that the production strain was the problem. Therefore, we will try the same procedure again but now in BL-21 E. coli.

We transformed electrocompetent BL-21 cells with Bba_K883401, which contains a PLRV Coat Protein encoding gene under the IPTG inducible promotor.

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