Team:Wageningen UR/Journal/week21

From 2012.igem.org

(Difference between revisions)
(→week 21: 17 September - 23 September)
Line 28: Line 28:
* colony PCR of the transformation from 17 September  
* colony PCR of the transformation from 17 September  
 +
 +
'''CCMV coil addition'''
 +
 +
----
 +
Frame shift in coil addition
 +
After recieving our samples from the sequencing, we saw that there was a frame shift in the sequence, leading to the faulty translation of the CCMV delta 25 coat protein, with an early stop codon.
 +
 +
{| class="wikitable" style="text-align: center"
 +
|- style="font-style: bold"
 +
|
 +
|faulty
 +
|intended
 +
|-
 +
|BP sequence
 +
| GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGATAGC
 +
GGCGTTGAAGGAGAAAATCGCAGCACTAAAAGAAAAGATAG
 +
CGGCGTTGAAGGAGCTTGGTGGTGGTTCTGGTGGTGGTGGT
 +
TCTGCTGCTGC'''TGT'''GTGGTCCAACCTGTTATTGTAGAACCC
 +
ATCGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCGG
 +
TTACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCCG
 +
AAGCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAAT
 +
GAGCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTAG
 +
AGTTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGCA
 +
CAGTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGCT
 +
GCCTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGAA
 +
AGATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGTA
 +
TAACCCTTGAACAACTCACCGCGGATTTAACGATCTACTTG
 +
TACAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGCA
 +
TTTGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCTT
 +
TCACTCCGGTGTAT
 +
 +
| GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGATAGC
 +
GGCGTTGAAGGAGAAAATCGCAGCACTAAAAGAAAAGATAG
 +
CGGCGTTGAAGGAGCTTGGTGGTGGTTCTGGTGGTGGTGGT
 +
TCTGCTGCTGC'''TGGT'''GTGGTCCAACCTGTTATTGTAGAACC
 +
CATCGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCG
 +
GTTACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCC
 +
GAAGCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAA
 +
TGAGCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTA
 +
GAGTTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGC
 +
ACAGTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGC
 +
TGCCTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGA
 +
AAGATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGT
 +
ATAACCCTTGAACAACTCACCGCGGATTTAACGATCTACTT
 +
GTACAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGC
 +
ATTTGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCT
 +
TTCACTCCGGTGTAT
 +
|-
 +
| primer FW1 FL-Delta26
 +
| TGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTGCTGC
 +
'''TGT''''GTGGTCCAACCTGTTATTGTAG
 +
| TGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTGCTGC
 +
'''TGGT'''GTGGTCCAACCTGTTATTGTAG
 +
 +
|-
 +
| translation product (AA)
 +
| MKIAALKEKIAALKEKIAALKELGGGSGGGGSAAAVW
 +
SNLLL
 +
| MKIAALKEKIAALKEKIAALKELGGGSGGGGSAAAGV
 +
VQPVIVEPIASGQGKAIKAWTGYSVSKWTASCAAAEAKV
 +
TSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTV
 +
KSCVTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKG
 +
ITLEQLTADLTIYLYSSAALTEGDVIVHLEVEHVRPTFD
 +
DSFTPVY
 +
|}
 +
 +
the new primer is ordered to still make this part available for the registry, but submission before the wiki deadline will not be possible anymore. however, we think that iGEM should not be the only reason to deliver bricks, and we intend to deliver all bricks that did not make it just in time, still after the iGEM deadlines.
----
----
[[https://2012.igem.org/Team:Wageningen_UR/Journal/week20 previous week]]
[[https://2012.igem.org/Team:Wageningen_UR/Journal/week20 previous week]]

Revision as of 10:20, 24 September 2012

week 21: 17 September - 23 September

Hepatitis B inside modification


  • Colony PCR of Hepatitis B core protein behing an IPTG promoter in pSB1C3 backbone with sequencing primers

-> the gel shows fragments of the expected size (about 1kb)


GFP modification

17 September


  • ligation of the PCR products from 14 September (mixing the duplo samples together) with pJET and transformation with JM109


18 September

  • colony PCR of the transformation from 17 September

CCMV coil addition

Frame shift in coil addition After recieving our samples from the sequencing, we saw that there was a frame shift in the sequence, leading to the faulty translation of the CCMV delta 25 coat protein, with an early stop codon.

faulty intended
BP sequence GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGATAGC

GGCGTTGAAGGAGAAAATCGCAGCACTAAAAGAAAAGATAG CGGCGTTGAAGGAGCTTGGTGGTGGTTCTGGTGGTGGTGGT TCTGCTGCTGCTGTGTGGTCCAACCTGTTATTGTAGAACCC ATCGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCGG TTACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCCG AAGCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAAT GAGCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTAG AGTTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGCA CAGTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGCT GCCTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGAA AGATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGTA TAACCCTTGAACAACTCACCGCGGATTTAACGATCTACTTG TACAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGCA TTTGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCTT TCACTCCGGTGTAT

GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGATAGC

GGCGTTGAAGGAGAAAATCGCAGCACTAAAAGAAAAGATAG CGGCGTTGAAGGAGCTTGGTGGTGGTTCTGGTGGTGGTGGT TCTGCTGCTGCTGGTGTGGTCCAACCTGTTATTGTAGAACC CATCGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCG GTTACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCC GAAGCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAA TGAGCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTA GAGTTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGC ACAGTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGC TGCCTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGA AAGATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGT ATAACCCTTGAACAACTCACCGCGGATTTAACGATCTACTT GTACAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGC ATTTGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCT TTCACTCCGGTGTAT

primer FW1 FL-Delta26 TGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTGCTGC

TGT'GTGGTCCAACCTGTTATTGTAG

TGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTGCTGC

TGGTGTGGTCCAACCTGTTATTGTAG

translation product (AA) MKIAALKEKIAALKEKIAALKELGGGSGGGGSAAAVW

SNLLL

MKIAALKEKIAALKEKIAALKELGGGSGGGGSAAAGV

VQPVIVEPIASGQGKAIKAWTGYSVSKWTASCAAAEAKV TSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTV KSCVTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKG ITLEQLTADLTIYLYSSAALTEGDVIVHLEVEHVRPTFD DSFTPVY

the new primer is ordered to still make this part available for the registry, but submission before the wiki deadline will not be possible anymore. however, we think that iGEM should not be the only reason to deliver bricks, and we intend to deliver all bricks that did not make it just in time, still after the iGEM deadlines.


[previous week]

Retrieved from "http://2012.igem.org/Team:Wageningen_UR/Journal/week21"