Difference between revisions of "Team:Humboldt Berlin/Notebook"
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</div> | </div> | ||
<div class="timeline"> | <div class="timeline"> | ||
| − | <div class="timeline-month tl-first-element" data-text="- Learning MoClo/Golden Gate Design <br> | + | <!-- synthesis --> |
| + | <div class="timeline-month tl-first-element" | ||
| + | data-text="- Learning MoClo/Golden Gate Design <br> | ||
- Research on PETase degradation <br> | - Research on PETase degradation <br> | ||
- Planning construction design for Golden Gate cloning using 10 different fusion sites according to Patron <br> | - Planning construction design for Golden Gate cloning using 10 different fusion sites according to Patron <br> | ||
| − | - Designing Primers for C. reinhardtii specified promotor PsaD containing BpiI recognition site with MoClo fusion sites and fusion sites compatible for L0 backbone. Primers containing mutation to delete BpiI site (Primer 5,6,11-13)." data-date="10/08 - 10/14" data-category="synthesis"> | + | - Designing Primers for C. reinhardtii specified promotor PsaD containing BpiI recognition site with MoClo fusion sites and fusion sites compatible for L0 backbone. Primers containing mutation to delete BpiI site (Primer 5,6,11-13)." |
| + | data-date="10/08 - 10/14" | ||
| + | data-category="synthesis"> | ||
October <br> 2018 | October <br> 2018 | ||
</div> | </div> | ||
| − | <div class="timeline-dot" data-text="- Research on C. reinhardtii cultivation <br> | + | <div class="timeline-dot" |
| + | data-text="- Research on C. reinhardtii cultivation <br> | ||
<h2>YFP</h2> | <h2>YFP</h2> | ||
| − | - Designing Primers for YFP with B5-B5 fusion sites taken from AG Hegemann plasmid p135 (Primer 7+8)" data-date="10/15 - 10/21" data-category="synthesis"> | + | - Designing Primers for YFP with B5-B5 fusion sites taken from AG Hegemann plasmid p135 (Primer 7+8)" |
| + | data-date="10/15 - 10/21" | ||
| + | data-category="synthesis"> | ||
</div> | </div> | ||
| − | <div class="timeline-dot" data-text="- Searching for gene templates <br> | + | <div class="timeline-dot" |
| + | data-text="- Searching for gene templates <br> | ||
<h2>chemical competent DH10B</h2> | <h2>chemical competent DH10B</h2> | ||
- production of chemical competent E. coli DH10B cells <br> | - production of chemical competent E. coli DH10B cells <br> | ||
| − | - PCR for YFP B5-B5 with primer 7+8" data-category="synthesis" data-date="10/22 - 10/28"> | + | - PCR for YFP B5-B5 with primer 7+8" |
| + | data-category="synthesis" | ||
| + | data-date="10/22 - 10/28" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/e/eb/T--Humboldt_Berlin--notebook_s_1022.png"> | ||
</div> | </div> | ||
| − | <div class="timeline-month" data-text="<h2>chemical competent DH10B</h2> | + | <div class="timeline-month" |
| + | data-text="<h2>chemical competent DH10B</h2> | ||
- checking the chemical competent DH10B cells → Transformation of plasmid containing RFP (Plasmid: pP51) (result/picture: red colonies grew → cells are competent! no cells on negative control) | - checking the chemical competent DH10B cells → Transformation of plasmid containing RFP (Plasmid: pP51) (result/picture: red colonies grew → cells are competent! no cells on negative control) | ||
<h2>ARS</h2> | <h2>ARS</h2> | ||
- Design of Arylsulfatase 1 (ARS) secretion signal as a B2-B2 part | - Design of Arylsulfatase 1 (ARS) secretion signal as a B2-B2 part | ||
<h2>GLE</h2> | <h2>GLE</h2> | ||
| − | - Design of Gametolysin (GLE) secretion signal as a B2-B2 part" data-date="10/29 - 11/04" data-category="synthesis" data-image="https://static.igem.org/mediawiki/2019/1/17/T--Humboldt_Berlin--Notebook-10-29.png"> | + | - Design of Gametolysin (GLE) secretion signal as a B2-B2 part" |
| + | data-date="10/29 - 11/04" | ||
| + | data-category="synthesis" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/1/17/T--Humboldt_Berlin--Notebook-10-29.png"> | ||
November | November | ||
| + | </div> | ||
| + | <div class="timeline-dot" | ||
| + | data-text="<h2>PsaD</h2> | ||
| + | - 11+12 → fragment 1 <br> | ||
| + | - 12+6 → fragment 2 <br> | ||
| + | - 13+5 → fragment 3 <br> | ||
| + | → result: PCR successful <br> | ||
| + | - Second PRC to combine PsaD fragments: <br> | ||
| + | fragment 2 + 3 → part 1 <br> | ||
| + | fragment 1 + 3 → part 2 <br> | ||
| + | → result: PCR successful" | ||
| + | data-category="synthesis" | ||
| + | data-date="11/05 - 11/11" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/9/9f/T--Humboldt_Berlin--notebook_s_115.png"> | ||
| + | </div> | ||
| + | <div class="timeline-dot" | ||
| + | data-text="<div class='two-columns'><div> | ||
| + | <h2>PsaD</h2> | ||
| + | - Third PRC to amplify PsaD from outer sites <br> | ||
| + | 1) Template: part 1; primer: 5+6<br> | ||
| + | 2) Template: Part 2; Primer: 5+6<br> | ||
| + | result: PCR was successful <br> | ||
| + | Clean-Up of PCR products <br> | ||
| + | Golden Gate Restriction/Ligation of PsaD PCR Products (PsaD A1-A3 and PsaD A1-B1) with Level 0 plasmid using <i>Bpi</i>I | ||
| + | <h2>YFP</h2> | ||
| + | Recognition of frameshift in YFP; new design of Primers for p51 from AG Hegemann <br> | ||
| + | - Primer 24+25 → YFP B5-B5 <br> | ||
| + | - Primer 26 + 27 → YFP B3-B4 <br> | ||
| + | <h2>RFP</h2> | ||
| + | - Designing Primers to amplify RFP containing <i>Bpi</i>I and <i>Bsa</i>I recognition site (Primer 39/40) to build a modulated L0-backbone | ||
| + | </div> | ||
| + | <div> | ||
| + | <h2>ARS1</h2> | ||
| + | - New design of ARS1 secretion signal as a B2-B2 part <br> | ||
| + | - Oligos 16 + 17 were ordered | ||
| + | <h2>GLE</h2> | ||
| + | - New design of GLE secretion signal as a B2-B2 part <br> | ||
| + | - Oligos 18 + 19 were ordered | ||
| + | <h2>SP20</h2> | ||
| + | -Design of SP20 secretion enhancement as a B5-B5 part<br> | ||
| + | - Oligos 20 + 21 were ordered | ||
| + | </div></div>" | ||
| + | data-category="synthesis" | ||
| + | data-date="11/12 - 11/18" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/8/8c/T--Humboldt_Berlin--notebook_s_1112_2.png"> | ||
| + | </div> | ||
| + | <div class="timeline-dot" | ||
| + | data-text="<h2>RFP</h2> | ||
| + | - Designing Primers to amplify RFP containing <i>Bpi</i>I and <i>Bsa</i>I recognition site (Primer 39/40) to build a modulated L0-backbone | ||
| + | <h2>PsaD</h2> | ||
| + | - Heatshock Transformation of L0-PsaD A1/A3 and L0-PsaD A1/B1 into heat-competent <i>E.coli</i> DH10B | ||
| + | Result/Picture: white colonies carrying L0-PsaD, blue colonies just L0 | ||
| + | YFP Golden Gate Restriction/Ligation of YFP B5-B5 and YFP B3-B4 into L0" | ||
| + | data-category="synthesis" | ||
| + | data-date="11/12 - 11/18" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/9/93/T--Humboldt_Berlin--notebook_s_1119.png"> | ||
| + | </div> | ||
| + | <div class="timeline-month" | ||
| + | data-text="<h2>PsaD</h2> | ||
| + | - overnight cultures of DH10B with L0-PsaD A1/A3 and L0-PsaD A1/B1 | ||
| + | <div style='text-indent:20px;'>- plasmid prep of ON-culture</div> | ||
| + | <div style='text-indent:20px;'>- Nano-Drob concentration</div> | ||
| + | - sequencing result: positive sequencing result of 1 clone each" | ||
| + | data-date="11/26 - 12/02" | ||
| + | data-category="synthesis"> | ||
| + | December | ||
| + | </div> | ||
| + | <div class="timeline-dot" | ||
| + | data-text="<div class='two-columns'><div> | ||
| + | <h2>RFP</h2> | ||
| + | - PCR to amplify RFP from pPH041 backbone using primers 39/40 <br> | ||
| + | - RFP will be selection marker on L0 backbone within the fusion site <br> | ||
| + | - restriction of RFP with <i>Bsa</i>I; restriction of pAGM9121 backbone with <i>Bsa</i>I <br> | ||
| + | - Ligation of pAGM9121 backbone with RFP using T4 ligase → L0-RFP (RFP ist NOT an insert, its inside the cloning site) <br> | ||
| + | - Heatshock transformation of L0-RFP into heat-competent <i>E.coli</i> DH10B | ||
| + | </div> | ||
| + | <div> | ||
| + | <h2>ARS1</h2> | ||
| + | - Oligoannealing of Oligos 16 + 17 for the creation of the ARS1 secretion signal with B2 fusion sites | ||
| + | <h2>GLE</h2> | ||
| + | - Oligoannealing of Oligos 18 + 19 for the creation of the GLE secretion signal with B2 fusion sites | ||
| + | <h2>SP20</h2> | ||
| + | - Oligoannealing of Oligos 20 + 21 for the creation of the SP20 secretion enhancement with B5 fusion sites | ||
| + | </div></div>" | ||
| + | data-category="synthesis" | ||
| + | data-date="12/03 - 12/09" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/3/3a/T--Humboldt_Berlin--notebook_s_1203.png"> | ||
| + | </div> | ||
| + | <div class="timeline-dot" | ||
| + | data-text="<div class='two-columns'><div> | ||
| + | <h2>L0-RFP</h2> | ||
| + | - positive sequencing result of L0-RFP containing required restriction and fusion sites | ||
| + | <h2>PsaD</h2> | ||
| + | - Golden Gate Restriction/Ligation of PsaD PCR Products (PsaD A1-A3 and PsaD A1-B1) with L0-RFP plasmid using <i>Bpi</i>I | ||
| + | <h2>ARS1</h2> | ||
| + | - Ligation of ARS1 into L0-RFP<br> | ||
| + | - Heatshock transformation of L0-ARS into heat-competent <i>E.coli</i> DH10B<br> | ||
| + | - 3 Over-night cultures were made and prepped and sent for sequencing | ||
| + | <h2>GLE</h2> | ||
| + | - Ligation of GLE into L0-RFP<br> | ||
| + | - Heatshock transformation of L0-GLE into heat-competent <i>E.coli</i> DH10B<br> | ||
| + | - 3 Over-night cultures were made and prepped and sent for sequencing | ||
| + | </div> | ||
| + | <div> | ||
| + | <h2>SP20</h2> | ||
| + | - Ligation of SP20 into L0-RFP<br> | ||
| + | - Heatshock transformation of L0-SP20 into heat-competent <i>E.coli</i> DH10B<br> | ||
| + | - 3 Over-night cultures were made and prepped and sent for sequencing | ||
| + | <h2>His-Tag</h2> | ||
| + | - Design of a 6xHis-Tag for detection, isolation and purification as a C-terminal tag; B5-B5 module<br> | ||
| + | - Oligonucleotides 47+48 were ordered | ||
| + | <h2>HA-Tag</h2> | ||
| + | - Design of a 3xHA-Tag for detection, isolation and purification as a C-terminal tag; B5-B5 module<br> | ||
| + | - Oligonucleotides 49+50 were ordered | ||
| + | </div></div>" | ||
| + | data-category="synthesis" | ||
| + | data-date="12/10 - 12/16" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/e/e1/T--Humboldt_Berlin--notebook_s_1210_1.png"> | ||
| + | </div> | ||
| + | <div class="timeline-dot" | ||
| + | data-text="<div class='two-columns'><div> | ||
| + | <h2>YFP</h2> | ||
| + | - Heatshock Transformation of L0-YFP B5-B5 and B3-B4 into heat-competent <i>E.coli</i> DH10B | ||
| + | 3 Over-night cultures were made and prepped and sent for sequencing | ||
| + | <h2>PsaD</h2> | ||
| + | - Heatshock Transformation of L0-PsaD A1/A3 and L0-PsaD A1/B1 into heat-competent <i>E.coli</i> DH10B <br> | ||
| + | - Result/Picture: white colonies carrying L0-PsaD, red colonies just L0-RFP | ||
| + | </div> | ||
| + | <div> | ||
| + | <h2>His-Tag</h2> | ||
| + | - Oligoannealing of 47+48 to produce the 6xHis-Tag with B5-B5 fusion sites<br> | ||
| + | - Ligation of the annealed product to L0-RFP | ||
| + | <h2>HA-Tag</h2> | ||
| + | - Oligoannealing of 49+50 to produce the 3xHA-Tag with B5-B5 fusion sites<br> | ||
| + | - Ligation of the annealed product to L0-RFP | ||
| + | </div></div>" | ||
| + | data-category="synthesis" | ||
| + | data-date="12/17 - 12/23" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/7/77/T--Humboldt_Berlin--notebook_s_1217_1.png"> | ||
| + | </div> | ||
| + | <div class="timeline-month" | ||
| + | data-text="<h2>YFP</h2> | ||
| + | - Positive sequencing results of L0-YFP B5-B5 and B3-B4 | ||
| + | <h2>ARS1</h2> | ||
| + | - Positive sequencing results of L0-ARS B2-B2 | ||
| + | <h2>GLE</h2> | ||
| + | - Positive sequencing results of L0-GLE B2-B2 | ||
| + | <h2>SP20</h2> | ||
| + | - Positive sequencing results of L0-SP20 B5-B5 | ||
| + | <h2>PsaD</h2> | ||
| + | - Positive sequencing results of L0-PsaD A1/A3 <br> | ||
| + | - negative sequencing results of L0-PsaD A1/B1" | ||
| + | data-date="12/31 - 01/06" | ||
| + | data-category="synthesis"> | ||
| + | January | ||
| + | </div> | ||
| + | <div class="timeline-dot" | ||
| + | data-text="<h2>His-Tag</h2> | ||
| + | - Failed Heatshock Transformation of L0-His-Tag into heat-competent <i>E.coli</i> DH10B (07.01.) <br> | ||
| + | - Repetition of Heatshock transformation (10.01.) <br> | ||
| + | - 3 Over-night cultures were made and prepped and sent for sequencing <br> | ||
| + | <h2>HA-Tag</h2> | ||
| + | Failed Heatshock Transformation of L0-HA-Tag into heat-competent <i>E.coli</i> DH10B (07.01.) <br> | ||
| + | Repetition of Heatshock transformation (10.01) <br> | ||
| + | 3 Over-night cultures were made and prepped and sent for sequencing" | ||
| + | data-category="synthesis" | ||
| + | data-date="01/07 - 01/13" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/5/5b/T--Humboldt_Berlin--notebook_s_1217_4.png"> | ||
| + | </div> | ||
| + | <div class="timeline-month" | ||
| + | data-text="<h2>His-Tag</h2> | ||
| + | - Oligoannealing of 47+55 to produce the 6xHis-Tag with B5-B5 fusion sites <br> | ||
| + | - Ligation of the annealed product to L0-RFP <br> | ||
| + | - Heatshock Transformation of L0-His-Tag into heat-competent <i>E.coli</i> DH10B <br> | ||
| + | - Colony PCR <br> | ||
| + | - 3 Over-night cultures were made and prepped and sent for sequencing" | ||
| + | data-date="01/28 - 02/03" | ||
| + | data-category="synthesis" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/c/ca/T--Humboldt_Berlin--notebook_s_0128_1.png"> | ||
| + | February | ||
</div> | </div> | ||
| − | <div class="timeline-dot" data-text="- YOYOOY <br> | + | <!-- human practice --> |
| + | <div class="timeline-dot" | ||
| + | data-text="- YOYOOY <br> | ||
<h2>YFP</h2> | <h2>YFP</h2> | ||
| − | - Designing Primers for YFP with B5-B5 fusion sites taken from AG Hegemann plasmid p135 (Primer 7+8)" data-date="10/15 - 10/21" data-category="human-practice"> | + | - Designing Primers for YFP with B5-B5 fusion sites taken from AG Hegemann plasmid p135 (Primer 7+8)" |
| + | data-date="10/15 - 10/21" | ||
| + | data-category="human-practice"> | ||
</div> | </div> | ||
| − | <div class="timeline-dot" data-text="- Searching for gene templates <br> | + | <div class="timeline-dot" |
| + | data-text="- Searching for gene templates <br> | ||
<h2>chemical competent DH10B</h2> | <h2>chemical competent DH10B</h2> | ||
- production of chemical competent E. coli DH10B cells <br> | - production of chemical competent E. coli DH10B cells <br> | ||
| − | - PCR for YFP B5-B5 with primer 7+8" data-category="human-practice" data-date="10/22 - 10/28"> | + | - PCR for YFP B5-B5 with primer 7+8" |
| + | data-category="human-practice" | ||
| + | data-date="10/22 - 10/28"> | ||
</div> | </div> | ||
| − | <div class="timeline-month" data-text="<h2>chemical competent DH10B</h2> | + | <div class="timeline-month" |
| + | data-text="<h2>chemical competent DH10B</h2> | ||
- checking the chemical competent DH10B cells → Transformation of plasmid containing RFP (Plasmid: pP51) (result/picture: red colonies grew → cells are competent! no cells on negative control) | - checking the chemical competent DH10B cells → Transformation of plasmid containing RFP (Plasmid: pP51) (result/picture: red colonies grew → cells are competent! no cells on negative control) | ||
<h2>ARS</h2> | <h2>ARS</h2> | ||
- Design of Arylsulfatase 1 (ARS) secretion signal as a B2-B2 part | - Design of Arylsulfatase 1 (ARS) secretion signal as a B2-B2 part | ||
<h2>GLE</h2> | <h2>GLE</h2> | ||
| − | - Design of Gametolysin (GLE) secretion signal as a B2-B2 part" data-date="10/29 - 11/04" data-category="human-practice" data-image="https://static.igem.org/mediawiki/2019/1/17/T--Humboldt_Berlin--Notebook-10-29.png"> | + | - Design of Gametolysin (GLE) secretion signal as a B2-B2 part" |
| + | data-date="10/29 - 11/04" | ||
| + | data-category="human-practice" | ||
| + | data-image="https://static.igem.org/mediawiki/2019/1/17/T--Humboldt_Berlin--Notebook-10-29.png"> | ||
November | November | ||
</div> | </div> | ||
Revision as of 06:30, 15 October 2019
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