From 2014.igem.org

(Difference between revisions)

Revision as of 17:50, 29 September 2014

X

Next ⇒

⇐ Prev

...

Lab notes

introduction

control

communication

cohesion

completion

function test

introduction :P introduction :P introduction :P introduction :P

introduction of labnote

樹狀圖 timeline 順序圖

under construction

lab note of circuit in the cohesion part

Working Circuit Assembling

1. Check IGEM standard part-K523013(trunk).B0015

IGEM kit transformation with DH5 competent cell
colony PCR check: pick up colony of transformation and do the PCR to check part length
plasmid PCR check: extract the plasmid of the correct length of part after checking colony PCR check, and use plasmid PCR to check length again
Digestion check: use enzyme to cut plasmid of correct length of PCR, to check restriction site
Second plate and liquid culture save: save bacteria containing correct plasmid in liquid culture and second plate

2. Get S. mutans competent signal peptide (CSP16) part

S. mutans genome extraction
Tag PCR to get CSP16 linear DNA and check length through electrophoresis: we use Tag PCR to test the condition of the most suitable condition of PCR
KOD PCR to get CSP16 linear DNA, check length through electrophoresis, and gel extraction to get CSP16 part: in the condition tested during Tag PCR, we use designed primer and KOD PCR to amplify and copy CSP16 linear DNA , and run electrophoresis and gel extraction to get CSP16 part
Digestion check: use enzyme to cut plasmid of correct length of PCR, to check restriction site
Second plate and liquid culture save: save bacteria containing correct plasmid in liquid culture and second plate

3. K523013(trunk) and CSP16 assembling

We use Tag PCR with designed primer to check suitable PCR condition
We conducted KOD PCR with designed primer to get K523013 assembled with CSP16 linear DNA and check length through electrophoresis, and gel extraction to get CSP16 plus K523013(trunk) part: in the condition tested during Tag PCR, we use designed primer and KOD PCR to assemble CSP16 linear DNA and K523013(trunk) , and run electrophoresis and gel extraction to get CSP16 assembled with K523013(trunk) part

4. K523013 assembled with CSP16 and BBa-B0015 assembling

K523013 assembled with CSP16 digestion: we use ECOR1 and SPET1 enzyme to digest K523013 assembledCSP16
BBa-B0015 digestion: we use ECOR1 and XBAL1 enzyme to digest BBa-B0015
K523013 assembled with CSP16and BBa-B0015 ligation: we use T4 ligase to ligase K523013 assembled with CSP16and BBa-B0015
transformation with DH5ɚ competent cell
After plasmid extraction of the ciucuit ,it ran PCR and electrophoresis to check length of circuit
digestion to check circuit length and restriction site
plasmid sequencing of the final circuit

Testing Circuit Assembling

1. Check IGEM standard part-E1010

IGEM kit transformation with DH5ɚ competent cell
colony PCR check: pick up colony of transformation and do the PCR to check part length
plasmid PCR check: extract the plasmid of the correct length of part after checking colony PCR check, and use plasmid PCR to check length again
Digestion check: use enzyme to cut plasmid of correct length of PCR, to check restriction site
Second plate and liquid culture save: save bacteria containing correct plasmid in liquid culture and second plate

2. K523013 + CSP16 and E1010 assembling

K523013 assembled with CSP16 digestion: we use ECOR1 and SPET1 enzyme to digest K523013 assembledCSP16
E1010 digestion: we use ECOR1 and XBAL1 enzyme to digest E1010
K523013 assembled with CSP16and E1010 ligation: we use T4 ligase to ligase K523013 assembled with CSP16and E1010
transformation with DH5 competent cell
plasmid extraction of the circuit and through PCR and electrophoresis to check length of circuit
digestion to check circuit length and restriction site of circuit
plasmid sequencing of the final circuit

3. K523013 + CSP16 + E1010 and B0015 assembling

K523013 assembled with CSP16 digestion: we use ECOR1 and SPET1 enzyme to digest K523013 assembledCSP16 and E1010
BBa-B0015 digestion: we use ECOR1 and XBAL1 enzyme to digest BBa-B0015
K523013 assembled with CSP16 assembled with E1010and BBa-B0015 ligation: we use T4 ligase to ligase K523013 assembled with CSP16 assembled with BBa-B0015
transformation with DH5 competent cell
plasmid extraction of the ciucuit and through PCR and electrophoresis to check length of circuit
digestion to check circuit length and restriction site of circuit
plasmid sequencing of the final circuit

lab note of circuit in the completion part

Parts from iGEM took kit

Out line

For each part to be used in testing circuit:
BBa_J23100 , BBa_E1010, BBa_B0015

circuit 圖

After transforming these plasmids into our competent cell, DH5a, we would take 3 steps to examine if we got the right product:

Colony PCR and electrophoresis: Directly picking up colonies on designated antibiotic plate and do colony PCR with iGEM provided primers (VF2&VR), and then do electrophoresis to check the length of plasmid to confirm if the kit had been correctly transformed into E. coli.
Plasmid PCR and electrophoresis: Colony PCR may not end up copying the right place, for there is extra DNA in the cell that is not the one of our interest. Therefore, we also check the length of the plasmid we which we transformed.
Digestion and electrophoresis: After checking the length, we use EcoR1/Spe1 and Xba1/Pst1, and electrophoresis, to see if our constructed plasmid can be successfully digested.

Process

We encounter several difficulties during the process:

In the transformation step, we couldn’t get transformation with the right PCR band at first. Even thought they do grow on the antibiotic plate, electrophoresis result doesn’t show any band. Therefore, we tried different ways trying to solve these problems.
First, we thought it was PCR problem, therefore, we tried colony PCR. However, it came out that it wasn’t the case.So we improve our transformation protocol. That is, after 1.5 hour of recovery in 37C incubator in 200ml LB, we centrifuge in 3.4 rpm for 20 seconds, and discard 150ml supernatant.
At the end, we result in putting concentrated transformed E.coli into our antibiotic plate. This way, we sequentially transformed several kits successfully.
In the checking step, we’ve have contaminated electrophoresis result after colony PCR. That is, we got band in the negative well. To find out the ingredient causing contamination, we do several negative control changing different packing of ingredients. And find the contamination of our 10X DreamTaq Buffer.

Result

Parts that needed extra PCR

Outline

Two parts needed extra primer in order to get them:
yebF, RFP, and M102 orf19

circuit 圖

And for each part, we do two kinds of PCR , one using Dream Tag, only to check the length to ensure we got our part. And then we used KOD enzyme, together with magnesium cation, to check the length, furthermore, do gel extraction to get the part. That’s because KOD enzyme leads a more accurate PCR.

For yebF, we designed primers to get the sequence we want yebF (its own ribosomal binding site included) from Escherichia coli MG1655.
Front Primer : tttctagagGGAGAAAAACATGAAAAAAAGAGGGGCGTT(with Xbal cutting site)
Reverse Primer : attctgcagcggccgctactagtACGCCGCTGATATTCCGCCA(with Spel and Pst1 cutting site)
The PCR product should be 389bp.
Because the need of recombinant protein, the RFP downstream of yebF need to be specially designed in order to leave scar with multiple of 3. E1010 was used as template, and primers with specially designed cutting site is made:
Front Primer: tttctagaGCTTCCTCCTCCGAAGACGTTATC
And normal iGEM reverse primer, VR

Process

While doing RFP PCR, we got extra band.
By adjusting PCR program, we finally got correct band.

Result

Construction of the circuit

Outline

Circuits needed to be constructed
J23119-(RBS)yebF-RFP-B0015
J23119_B0034_RFP-B0015
J23119-yebF-B0015
J23119-yebF-orf19-B0015
By repeatedly using back insert, we tried to achieve the final construction.We planned to constructed our first circuit by following steps:

J23119(SP)+yebF(XP)
J23119+yebF(SP)+RFP(XP)
J23119+yebF+RFP(SP)+B0015(XP)

Process

During circuit construction, we encountered several problems:
First, we the enzyme that we own are from different companies. Our digestion enzyme Pst1 is from Fast Digestion, requiring FD buffer, while the Spel enzyme is from NEB, requiring the addition of NEB buffer 4 and BSA, or smart cut buffer.
Also, because that one of the part result from cutting is so small that it’s invisible in electrophoresis, we can only check if our product had been successfully cut by transformation. We also did electrophoresis, though, to check if the band would be good enough to do extraction. Finally, by doing checking transformation product after ligation, we can fully sure that we had the right digestion cut.
Our first ligation result shows that using FD buffer is absolutely impossible if we want to digest with Spel and Pst1. As shown on the graph, it might just stick back to the original plasmid.

@@ Line 112: / Line 112: @@
      </ol>
 <h2>Testing Circuit Assembling</h2>
+<p><b>1.	Check IGEM standard part-E1010</b></p>
+    <ol>
+       <li>IGEM kit transformation with DH5ɚ competent cell</li>
+       <li>colony PCR check: pick up colony of transformation and do the PCR to check part length</li>
+       <li>plasmid PCR check: extract the plasmid of the correct length of part after checking colony PCR check, and use plasmid PCR to check length again</li>
+       <li>Digestion check: use enzyme to cut plasmid of correct length of PCR, to check restriction site</li>
+       <li>Second plate and liquid culture save: save bacteria containing correct plasmid in liquid culture and second plate</li>
+       <img src=''>
+    </ol>
+<p><b>2.	K523013 + CSP16 and E1010 assembling</b></p>
+    <ol>
+       <li>K523013 assembled with CSP16 digestion: we use ECOR1 and SPET1 enzyme to digest K523013 assembledCSP16</li>
+       <li>E1010 digestion: we use ECOR1 and XBAL1 enzyme to digest E1010</li>
+       <li>K523013 assembled with CSP16and E1010 ligation: we use T4 ligase to ligase K523013 assembled with CSP16and E1010</li>
+       <li>transformation with DH5 competent cell </li>
+       <li>plasmid extraction of the circuit and through PCR and electrophoresis to check length of circuit</li>
+       <li>digestion to check circuit length and restriction site of circuit</li>
+       <li>plasmid sequencing of the final circuit</li>
+       <img src=''>
+    </ol>
+<p><b>3.	K523013 + CSP16 + E1010 and B0015 assembling</b></p>
+    <ol>
+       <li>K523013 assembled with CSP16 digestion: we use ECOR1 and SPET1 enzyme to digest K523013 assembledCSP16 and E1010</li>
+       <li>BBa-B0015 digestion: we use ECOR1 and XBAL1 enzyme to digest BBa-B0015</li>
+       <li>K523013 assembled with CSP16 assembled with E1010and BBa-B0015 ligation: we use T4 ligase to ligase K523013 assembled with CSP16 assembled with BBa-B0015</li>
+       <li>transformation with DH5 competent cell</li>
+       <li>plasmid extraction of the ciucuit and through PCR and electrophoresis to check length of circuit</li>
+       <li>digestion to check circuit length and restriction site of circuit</li>
+       <li>plasmid sequencing of the final circuit</li>
+       <img src=''>
+       <img src=''>
+    </ol>
           </div>
 <!----------------------------------------------------------->

Team:NYMU-Taipei/notebook/labnotes

From 2014.igem.org

Revision as of 17:50, 29 September 2014

introduction :P introduction :P introduction :P introduction :P

lab note of circuit in the control part

experiment1 date: 0707~0712

experiment1 date: 0707~0712

experiment1 date: 0707~0712

lab note of circuit in the communication part

experiment1 date: 0707~0712

experiment1 date: 0707~0712

experiment1 date: 0707~0712

lab note of circuit in the cohesion part

Working Circuit Assembling

Testing Circuit Assembling

lab note of circuit in the completion part

Parts from iGEM took kit

Out line

Process

Result

Parts that needed extra PCR

Outline

Process

Result

Construction of the circuit

Outline

Process

lab note of function test in phage

experiment1 date: 0707~0712

experiment1 date: 0707~0712

experiment1 date: 0707~0712