Week Beginning: 20th July 2009

Experiments

Conclusion

Monday

No experiments were undertaken

The aim of the week was to become acquainted with the protocols.

Tuesday

No experiments were undertaken

Wednesday

Pure Yield Plasmid Prep: practice session

Thursday

No experiments were undertaken

Friday

DNA Gel Electrophoresis: practice session

Week Beginning: 27th July 2009

Experiments

Conclusion

Monday

Bio brick elution and transformation

Experiments were carried out to become acquainted with the protocols.

Tuesday

Double digestion protocol

Wednesday

Gel extraction and purification

Thursday

High fidelity PCR

Friday

No experiments were undertaken

Week Beginning: 3rd Aug 2009

Experiments

Conclusion

Monday

No experiments undertaken

The aim of the week was to start transforming the necessary biobricks needed for the project.

Tuesday

Biobrick DNA Elution and Transformation:

E0040 K145270 C0012 I719005 R0071 K084009 C0161 K091100

Wednesday

Plasmid Prep (using transformed biobricks prepared yesterday – except C0161)

DNA Elution and Transformation
C0178 K081008 C0179 K091146 C0062 K091107 C0161

Thursday

Plasmid Prep (using biobricks transformed yesterday)

Friday

Repeat Biobrick transformations:
C0161

Week Beginning: 10th August 2009

Experiments

Conclusion

Monday

Culture overnight of transformed parts

B0015-Failed B0034 –Failed J23119-Failed C0161-Failed R0062 C0170 C0171 R0079

The main aim of this week was to grow up any bio-bricks that may be required. Several parts failed and so had to be repeated. There are many reasons for the failure, one being the use of the wrong cells and transformations failing.

Tuesday

Plasmid Maxiprep of the overnight cultures

Wednesday

Culture overnight of transformed parts

C0161 B0034 B0015 J23119 R0010 R0011

Thursday

Plasmid maxiprep of the overnight cultures

Friday

Digestion of parts for Quality control

Week Beginning: 17th Aug 2009

Experiments

Conclusion

Monday

No experiments were undertaken

The aim of the week was to start ligating biobrick parts together. The aim for next week is to check that the ligations have been successful.

Tuesday

Digestion of parts for quality control.

Polymerase Chain Reaction:

C0012 C0170 C0171 C0179 C0161 C0178

Wednesday

Polymerase Chain Reaction: the reaction from yesterday failed hence the PCR was repeated

Starter Culture and Maxi Prep:
mCherry mBanana GFP

Thursday

Double Digestion of PCR samples:
C0012 C0171 C0170 C0179 C0161 C0178

PCR Purification

Friday

Ligation of Biobrick parts- all the following parts were ligated with B0034:

C0171 C0179 C0012 C0178 C0170 C0062 C0161

Week Beginning: 24th August 2009

Experiments

Conclusion

Monday

Ligation of B0034 with C0178, C0062, E0040, C0171, C0179, C0170, C0012, C0161, E0040

The main aim of this week was to get our first ligations to work, however this wasn’t the case and so after the ligations were repeated and failed again, troubleshooting began.

Tuesday

Transformation of B0034
with biobrick parts:
C0178, C0062, E0040, C0171, C0179, C0170, C0012, C0161, E0040

Wednesday

Colony PCR of ligated parts
FAILED

PCR of Ptac from Pmal vector, which introduces prefix and suffix followed by a double digestion of pTAC and RBS.

Thursday

Repeat of Colony PCR, which had previously failed.

Friday

PCR of biobrick inserts

C0178, C0062, E0040, C0171, C0179, C0170, C0012, C0161, E0040
Required due to previous failure using up stocks.

Week Beginning: 31st Aug 2009

Experiments

Conclusion

Monday

No experiments were done today
(Bank Holiday)

The aim of this week was to continue trying to get our first ligations to work, this wasn’t successful the digestion was found not to be complete and so this is thought to be one of the reasons behind the failed ligations. The next set of Digestions will be done overnight instead of the usual 2 hours.

Tuesday

Repeat of Colony PCR of: pTac – B0034

DNA Double Digestion: (pTac – B0034)

Ligation: (pTac – B0034)

Wednesday

Colony PCR Control:
Method as per 28/08 but both B0034 and pTac colonies were diluted in 100μl.

Single Digestion of biobricks: single digestion was performed as it was discovered that one of the enzymes was not working efficiently.

C0171 C0179 C0012 C0178 C0170 C0062 C0161 E0040 B0034

DNA Transformation: pTac and B0034
Culture of C0161, pTac –B0034 and B0034

Thursday

Mini Prep
C0161 B0034-A B0034-B

Friday

Second Single Digestion:

C0171 C0170 C0012 C0179 E0040 0034
Biobricks (C0178, C0062, C0161 were transformed again)

Week Beginning: 7th Sept 2009

Experiments

Conclusion

Monday

PCR of Pmal to produce Ptac
Purification Gel of C0171, C0012, C0170, C0179, E0040, B0034. B0034 was treated with SAP.
C0171, C0012 and C0170 was ligated to B0034

TSAP (thermo sensitive alkaline phosphatase) was used on the plasmid to dephosphorylate the plasmid so it cannot religate. Digestions with PstI were done for 8 hours and every other digestion was run for 3 hours. Ligations are now ran overnight at 4C.

Tuesday

Digestion of Ptac with EcoRI and XbaI and RBS with XbaL and Spel RBS was treated with SAP
Ligation of Ptac to RBS
Culture overnight of biobrick parts C0161, C0178 and C0062 as these were shown to be dodgy parts.

Wednesday

A maxiprep was performed on the parts, which were previously cultured overnight. Digestion of the parts with Pstl to check that the parts were correct.

Thursday

PCR of C0161, C0178, C0062 which didn’t work

Friday

PCR of Gfp-LVA, which didn’t work.

Week Beginning: 14th Sept 2009

Experiments

Conclusion

Monday

Transformation of C0170, C0012 and pTac to RBS
PCR of GFP-LVA with different primers and it worked.

The ligations of Ptac didn’t work with the 3-hour digestion and TA cloning was inconclusive, due to a lack of resources. Parts, which were shown to be dodgy, were re-grown and checked for quality control.

Tuesday

Colony PCR of the ligated parts
Culture overnight of C0161, C0178, C0062.

Wednesday

Digestion of Biobricks pLux, STOP and LacI.followed by an overnight ligation of the parts
TA cloning of Ptac.
Maxiprep of the cultures from previous day.

Thursday

Transformation of the ligated parts
PCR of GFP LVA suffix with different primers, which was successful again.

Friday

Colony PCR of the ligated parts
PCR of C0161, C0178 that worked.

Week Beginning: 21st Sept 2009

Experiments

Conclusion

Monday

Diluted colony PCR of the C0012-B0015, R0062-C0012.
Overnight digestion of Ptac.
Ligation of Gfp-LVA to plasmid.

TA cloning was performed on Gfp-LVA to check the prefix and the suffix was digested properly.  Ptac also had a successful TA cloned vector. This means that the work to check for complete digestion can begin. The main focus of this week was to finish any wiki work as this is the last week for the iGEM students.

Tuesday

PCR of Ptac from Pmal
Overnight ligation for TA cloning.
Transformation of previously ligated part.

Wednesday

Digestion of C0012 and C0179
Gel extraction was performed, yielding too low concentrations for a ligation to occur.

Thursday

Repeat of the previous day’s experiment followed by ligation overnight.

Culture of the Gfp which was TA cloned

Friday

Maxiprep of the cultured Gfp. This failed as the DNA was sheared.

Week Beginning: 28th Sept 2009

Experiments

Conclusion

Monday

Culture of the TA cloned Ptac
Transformation of C0012-C0179 and Ptac-RBS

The transformations failed from the beginning of the week and the conclusion of the Gfp after ligation wasn’t reached until the next week.

Tuesday

The Ptac and Gfp-LVA which was TA cloned was digested overnight.
Colony PCR was performed on the ligations and was shown to have failed.

Wednesday

A gel was run on the Ptac and was shown to have digested improperly.  Gel purification was performed on Gfp-LVA

Thursday

Digestion of Ptac overnight and further purification of GFP-LVA.

Friday

Ligations of Ptac and Gfp-LVA with PSB1A3.

Week Beginning: 5th Oct 2009

Experiments

Conclusion

Monday

Digestion of Ptac with EcoRI HF and SpeI
Ptac Gel purification
TSAP with PSB1A3 ES digests.
Ligation of GFP-LVA (ES) into PSB1A3
Transformation of previous Ligation
More plates made up

Finally there was a successful ligation. The conditions, which seemed to work, were 18 hours at 16°C. Work is now going to be focussed on showing the functionality of the Ptac.

Tuesday

No colonies grew on the plates, suspected that the T4 Ligase was improperly killed.

Wednesday

Ligation of Ptac and GFP-LVA with PSB1A3

Thursday

Transformations performed on the previous ligations

Friday

Colonies observed on plates. Colony PCR performed on both, GFP-LVA discovered not to work.
Ptac, all colonies picked were successful

Week Beginning: 12th Oct 2009

Experiments

Conclusion

Monday

Grew up Ptac colonies, 3x Minipreps were performed. The yield was very low, so a culture for a maxiprep was then put on.
Digestion of RBS, GFP and B0015.

The focus of this week was to build a construct, which could prove the functionality of the Ptac part.

Tuesday

Ptac was Maxiprep’d. The yield again was very low, we think this was due to the introduction of the insert causing the plasmid to become low copy.
Gel purification of the previously digested parts.

Wednesday

Single Digestion of Ptac was performed.

Thursday

After gel purification of the Ptac, the yield was very low and so a double digestion of Ptac was performed.

Friday

Gel purification of Ptac part.
TSAP of the E0040 vector.
Ligation of Ptac and E0040.

Week Beginning: 17th Oct 2009

Experiments

Conclusion

Saturday

Transformation of the previously ligated parts.
Double digestion of the TA cloned Ptac.

The focus on this week was to continue trying to build a part, which will show functionality, and to finish off everything ready for the jamboree.

Sunday

Gel purification of the Ptac followed by ligation to the E0040 vector.
Colonies were observed, however after colony PCR, the ligation was shown to have failed.

Monday

Cultures of a fluorescent protein were grown up with the Ptac inserted.

Tuesday

Fluorescence studies were conducted on the Ptac cultures.
 Last minute touches to the Wiki, Poster and presentation.

Wednesday

DEADLINE FOR WIKI FREEZE