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Team:Wageningen UR/Journal/week21
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| - | '' | + | ''19 September'' |
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| + | * colony PCR of the transformation from 17 September (with pJET sequencing primers) | ||
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| + | [[File:Colony PCR 19Sept.jpg|frame|center|Figure 1: Colony PCR 19 September]] | ||
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| + | -> a lot of colonies show to have the correct insert size (around 950bp) | ||
| + | |||
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| + | ''20 September'' | ||
| + | |||
| + | * miniprep 2 colonies of the GFP-coil and 2 colonies of the GFP-coil with a His tag transformants from the Colony PCR | ||
| + | |||
| + | * Digestion check of these minipreps | ||
| + | -> shows the expected fragment sizes for all the samples | ||
| + | |||
| + | * sent 1 sample of the GFP-coil and 1 sample of the GFP-coil with the His tag for sequencing | ||
| + | |||
| + | * Digestion and Ligation of both GFP-coil constructs in a pSB1C3 backbone as well as in Bba_J04500 (behind an IPTG induced promoter) | ||
| - | |||
'''CCMV coil addition''' | '''CCMV coil addition''' | ||
Revision as of 07:24, 25 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
19 September
- colony PCR of the transformation from 17 September (with pJET sequencing primers)
-> a lot of colonies show to have the correct insert size (around 950bp)
20 September
- miniprep 2 colonies of the GFP-coil and 2 colonies of the GFP-coil with a His tag transformants from the Colony PCR
- Digestion check of these minipreps
-> shows the expected fragment sizes for all the samples
- sent 1 sample of the GFP-coil and 1 sample of the GFP-coil with the His tag for sequencing
- Digestion and Ligation of both GFP-coil constructs in a pSB1C3 backbone as well as in Bba_J04500 (behind an IPTG induced promoter)
CCMV coil addition
Frame shift in coil addition After receiving 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 |
GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGATAGCGG CGTTGAAGGAGAAAATCGCAGCACTAAAAGAAAAGATAGCG GCGTTGAAGGAGCTTGGTGGTGGTTCTGGTGGTGGTGGTTC TGCTGCTGCTGTGTGGTCCAACCTGTTATTGTAGAACCCAT CGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCGGTT ACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCCGAA GCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAATGA GCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTAGAG TTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGCACA GTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGCTGC CTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGAAAG ATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGTATA ACCCTTGAACAACTCACCGCGGATTTAACGATCTACTTGTA CAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGCATT TGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCTTTC ACTCCGGTGTAT |
GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGATAGCGG CGTTGAAGGAGAAAATCGCAGCACTAAAAGAAAAGATAGCG GCGTTGAAGGAGCTTGGTGGTGGTTCTGGTGGTGGTGGTTC TGCTGCTGCTGGTGTGGTCCAACCTGTTATTGTAGAACCCA TCGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCGGT TACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCCGA AGCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAATG AGCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTAGA GTTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGCAC AGTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGCTG CCTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGAAA GATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGTAT AACCCTTGAACAACTCACCGCGGATTTAACGATCTACTTGT ACAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGCAT TTGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCTTT CACTCCGGTGTAT |
| Primer FW1 FL-Delta26 |
TGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTGCTGC TGTGTGGTCCAACCTGTTATTGTAG | TGGTGGTGGTTCTGGTGGTGGTGGTTCTGCTGCTGC
TGGTGTGGTCCAACCTGTTATTGTAG |
| Translation product (AA) |
MKIAALKEKIAALKEKIAALKELGGGSGGGGSAAAVWSNLLL |
MKIAALKEKIAALKEKIAALKELGGGSGGGGSAAAGVVQPVI VEPIASGQGKAIKAWTGYSVSKWTASCAAAEAKVTSAITISL PNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSCVTETQTTA AASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYL YSSAALTEGDVIVHLEVEHVRPTFDDSFTPVY |
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.
