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Team:British Columbia/Notebook/Caffeine
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'''Experimentor :''' Liz Geum | '''Experimentor :''' Liz Geum | ||
<br> '''Aim : ''' To amplify the region between VF2 and VR to confirm the presence of 3kb insert in the vector. | <br> '''Aim : ''' To amplify the region between VF2 and VR to confirm the presence of 3kb insert in the vector. | ||
| - | <br>'''Results :''' 20 white colonies were picked after GFP screening. All colonies showed small bands of ~200 base pairs, and no 3kb band was present. | + | <br>'''Results :''' 20 white colonies were picked after GFP screening. All colonies showed small bands of ~200 base pairs, and no 3kb band was present. Also, the no-insert control plate showed just as many white colonies as the other plate. We concluded that there was a very high self-ligation at X and S cutsites in the vector, which outruns the 3kb stitched part to be inserted into the vector. Therefore, we decided to design new primers that will add the additional PstI cutsite at 3' end of our PCR 2 products. |
| + | |||
| + | ===Designing new primers for PCR 3=== | ||
| + | '''Experimentor :''' Ray Socha, Liz Geum, Grace Yi | ||
| + | <br>'''Aim :''' To design new primers that will add additional PstI cutsite at 3' end of PCR 2 products to avoid working with X/S cutsites. | ||
| + | <br>'''Results :''' | ||
===Restriction Digest with PCR 1 products=== | ===Restriction Digest with PCR 1 products=== | ||
Revision as of 01:10, 28 September 2013
iGEM Home
Caffeine Synthesis Notebook
Background Information: Overview of Main Reactions
"PCR 1": to remove the yeast consensus sequence of the 2013 Distribution Kit parts involved in caffeine synthesis
"PCR 2": to add new cut sites for PCR stitching
"PCR 3": to add a PstI cut site at the 3' end of the PCR 2 products
June 20th
Making Chemically Competent E.coli Cells
Experimenters: Joe Ho, Dan Korvin, Grace Yi
Aim: To make heat-shock competent cells for upcoming transformations
Results: The 1.5mL microcentrifuge tubes containing 100uL of cells were labelled "HS" on the lid and stored in the -80º Freezer #3. These will be tested for competency shortly.
June 22th
Ordering Primers for PCR 1
Experimenters: Grace Yi, Liz Geum, Ray Socha (advisor)
Aim: To design and order primers to modify the TU Munich caffeine biosynthesis BioBricks we got in the Distribution Kit.
Results: These BioBricks are found in wells 17H, 17J, and 17L on plate 2 and are parts BBa_K801070, BBa_K801071, BBa_K801072. Sequences were analyzed and a single set of primers was created to amplify all three genes after finding the 5' and 3' ends were mostly conserved. The latter was assumed to be so because of the strep tag the BioBrick creators incorporated.
June 27th
Transformation of parts BBa_K801070, BBa_K801071, BBa_K801072 (2013 Kit, Plate 2, Wells 17H, 17J, 17L)
Experimenters: Grace Yi, Liz Geum
Aim: To transform the TU Munich caffeine biosynthesis BioBricks from the Distribution Kit into E. coli chemically competent cells. This was also to test the cells' competency.
Results: Eluted DNA from wells 17H, 17J, and 17L on plate 2 were transformed via heat-shock into the chemically competent cells made earlier. These were plated on LB agar/chloramphenicol plates and incubated overnight at 37ºC. Transformation efficiency was found to be very low - two colonies per plate after more than 18 hours at 37ºC.
June 29th
Miniprep of parts 17H, 17J, 17L
Experimenters: Grace Yi, Liz Geum
Aim: To isolate the caffeine synthesis Biobrick plasmids we will use as template for downstream processing.
Results: Overnight cultures of LB and chloramphenicol were inoculated last night with colonies from the July 27 transformations. The three parts were miniprepped using the Qiagen Spin Miniprep Kit, with the following DNA concentrations:
H : 154.3 ng/µl
J : 323.8 ng/µl
L : 117.3 ng/µl
July 4
Confirmation PCR of miniprepped products
Experimentor : Grace Yi, Liz Geum
Aim : To confirm the presence of an insert in the backbone of miniprepped plasmids with VF2 and VR
Results : Gel confirmation of the PCR products showed a band around 1kb, our expected gene size, for one of the genes only (17J). The other samples did not show any bands. Because the transformation efficiency was very low, unsuccessful transformation was thought to be causing the issues. We decided to retry transformation of the kit parts into "tried and true" cells, and to discard today's plasmids if the new transformation gave different results.
July 5
Confirmation PCR of parts 17H, 17J, 17L
Experimentor : Liz Geum
Aim : To confirm our parts of interest (17H/J/L) contained the desired insert by running PCR on DNA from the Distribution Kit
Results : We ran a confirmation PCR with primers VF2 and VR, this time with 1 uL of eluted DNA from the distribution kit as template. Even with this low concentration of template, amplification appeared successful for one part. A bright band was seen for 17J and no bands for the other two, which mirrored yesterday's PCR results. As our project depended largely on the manipulation of the inserts in each part, we will be sequencing the plasmids we transformed today once colonies form and are miniprepped.
Primer Design
Experimentor : Grace Yi, Liz Geum, Ray Socha
Aim : To design and order primers to remove the "TCACA" yeast consensus sequence found in the TU Munich Biobrick parts
Results : Our goal was to replace this sequence with a bacterial ribosome-binding site. This round of PCR was named "PCR 1" to indicate downstream PCR reactions that would use these products as template.
July 7
Transformation of parts BBa_K801070, BBa_K801071, BBa_K801072 (2013 Kit, Plate 2, Wells 17H, 17J, 17L)
Experimentor : Grace Yi
Aim : To transform the three Biobricks containing caffeine biosynthesis genes. This is our second attempt at doing this.
Results : We acquired aliquots of tested chemically competent cells from Ray, our grad advisor, and re-tried the transformation of the distribution kit parts. We will find out tomorrow if it was successful. (Note: these three BioBricks will now be referred to as 17H, 17J, and 17L for convenience.)
July 8
Preparing Chemically Competent Cells
Experimentor : Joe Ho, Dan Korvin, Grace Yi
Aim : To prepare chemically competent cells to replace the first batch, which was found to be inefficient.
Results : Transformation of 17H/J/L with Ray's competent cells was found to be highly efficient, indicating our own heat-shock competent cells (which showed a very low transformation efficiency with the same plasmids) were faulty. We received the protocol used to prepare these cells, and a new stock was made today.
July 9
Miniprep of 17H, 17J, 17L
Experimentor : Liz Geum
Aim : To acquire 17H, 17J, 17L from 5-mL overnight cultures inoculated yesterday
Results : Cultures of 17H/J/L, which were grown in 5 mL of LB and Chlor overnight, were miniprepped. (The colonies picked were from transformations done on July 7.) With the products, we did the three following things.
- did a restriction digest with EcoRI and PstI to confirm the presence of an insert
- prepared each plasmid for sequencing to confirm the sequence of the insert (and the presence of the yeast consensus sequence, which we hoped to remove)
- PCR with primers designed to remove the yeast consensus sequence (TCACA): PCR 1
Restriction Digest with EcoRI and PstI
Experimentor : Liz Geum
Aim : To confirm the presence of an insert of the correct size
Results : The confirmation gel for the restriction digest showed there was an insert slightly larger than 1kb for each of the genes, which was desired. The band at 2kb was known to be pSB1C3.
Preparation of Samples for Sequencing
Experimentor : Grace Yi
Aim : To prepare and send newly-miniprepped 17H, 17J, and 17L to Genewiz for sequencing.
Results : Primers, VF2 and VR, were pre-mixed.
PCR 1: Removing the Yeast Consensus Sequence
Experimentor : Grace Yi
Aim : To remove the yeast consensus sequence, TCACA, from the 5' end of 17H, 17J, 17L
Results : PCR did not appear to work. Higher molecular weight smears were seen at around 2.5kb, indicating the template was not amplified, likely because there was too much of it. Around 200ng of plasmid template was used, annealing temperature was the lower primer Tm, and no DMSO was used. We will try this PCR again with much less template (10-50 ng per 50 uL reaction), as per NEB's suggestions, and fewer cycles.
July 11
PCR 1: Attempt #2
Experimentor : Grace Yi
Aim : To remove the yeast consensus sequence, TCACA, from the 5' end of 17H, 17J, 17L
Results : PCR 1 was retried for all three parts, with the amount of template reduced to around 30 ng per 50uL reaction. Phusion HF (High Fidelity) buffer was used, in the absence of DMSO. Thermocycling conditions were not changed, except the reduction of number of cycles from 30 to 20. Parts J and L were successfully amplified this time, with bands seen at around 1100-1200 bases on the agarose gel. However, nothing was seen for part H. One option is to try adding DMSO to a final concentration of 3% to hopefully allow amplification of part H.
Restriction Digest with XbaI and SpeI
Experimentor : Liz Geum
Aim : To cut insert and vector in preparation for ligation
Results : The amplified products for J and L, as well as pSB1C3 with GFP between the X and S cut sites, were digested with restriction enzymes XbaI and SpeI. We will be ligating J and L into the pSB1C3 tomorrow.
July 12
Designing Primers for "Stitching" the Caffeine Genes
Experimentor : Grace Yi, Liz Geum, Ray Socha
Aim : To design and order primers to "stitch" the caffeine genes together
Results : With Ray, we designed and ordered the "stitching" primers that would allow us to assemble our Biobricks easily, by using restriction enzyme cut sites other than the four standard ones. Once digested, two or three genes could be ligated together in a single ligation. This follows the decision to combine the three available (from the kit) caffeine biosynthesis genes and Biobrick them behind a promoter and ribosome-binding site (RBS). We included a bacterial ribosome-binding site in each of the forward primers to allow transcription when each gene is also Biobricked individually. This round of PCR reactions was named "PCR 2."
PCR 2 would ideally yield the following products:
- Part H: ---XbaI---gene H---SpeI---NcoI---
- Part J: ---NcoI---XbaI---gene J---SpeI---BamHI---
- Part L: ---BamHI---XbaI---gene L---SpeI---
Individually and ligated together, these products could be digested by XbaI and SpeI and inserted into a Biobrick vector cut with X and S.
July 15
PCR 1: Attempt #3 on part 17H
Experimentor : Grace Yi
Aim : To design and order primers to "stitch" the caffeine genes together
Results : PCR 1 was re-tried for part H, this time with the addition of DMSO. This was done in duplicate, to be able to compare the Phusion HF and GC buffers. PCR was successful, with bands seen at 1100 bases for both reactions. It appeared using either buffer would yield the same results, so we combined the two products and decided to use the HF buffer for consistency.
Restriction Digest with XbaI and SpeI
Experimentor : Liz Geum
Aim : To digest 17H, 17J, and 17L with XbaI and SpeI
Results : About 300 ng of today's PCR 1 product was cut with XbaI and SpeI. A larger amount (500 ng) of PCR 1 product for parts J and L (from July 11) were cut again with XbaI and SpeI, as a gel of the previous digest products showed very faint bands at the size of the insert.
July 17
Optimizing PCR 2
Experimentor : Grace Yi, Liz Geum
Aim : to optimize PCR 2 with stitching primers
Results : 3% DMSO and 59°C annealing temperature were optimal conditions for PCR 2. All three genes showed amplification at 1kb.
July 18
Restriction digest of PCR 2 products
Experimentor : Grace Yi, Liz Geum
Aim : Restriction digest PCR 2 with X and S cutsites for cloning
Results : The PCR 2 digests were run on gel and purified. Purified PCR 2 digest products were nanodropped with the following concentrations
H : 7.9 ng/µl
J : 12.4 ng/µl
L : 10.9 ng/µl
July 19
Ligation and transformation of PCR 2
Experimentor : Grace Yi, Liz Geum
Aim : To ligate PCR 2 digest products into empty pSB1C3 and transform into chemically competent cells.
Results : No insert ligation was set up as a control. Ligations reaction was left at room temperature for 3 hours, then heat inactivated. Competent cells were transformed.
July 22
Overnight cultures
Experimentor : Grace Yi, Liz Geum
Aim : To set up overnight culture for miniprep
Results : The no-insert control had many colonies, indicating high background re-ligation of X and S cutsites. The other three plates were filled with colonies as well. After GFP screening, 5 white colonies from each plate were inoculated.
July 23
Miniprep of PCR 2 transformation
Experimentor : Grace Yi, Liz Geum
Aim : To miniprep the colonies from PCR 2 transformation
Results : Overnight cultures were miniprepped with the following concentrations :
H1 : 127.2 ng/ µl
H2 : 103.5 ng/µl
J1 : 234.0 ng/µl
J2 : 140.3 ng/ µl
L1 : 128.0 ng/µl
L2 : 173.9 ng/ µl
The plasmids were sent in for sequencing with VF2 and VR.
July 24
Stitching PCR 2 products for cassette assembly
Experimentor : Grace Yi, Liz Geum
Aim : To digest the PCR 2 products with stitching enzymes and ligate for cassette assembly
Results : PCR 2 products were cut with the following the following enzymes :
H : NcoI
J : NcoI and BamHI
L : BamHI
60ng of each gene was used per 20ul ligation, and ligation was left at room temperature overnight.
July 25
Confirming the Presence of the Three-Gene Ligated Product
Experimentor: Grace Yi, Liz Geum
Aim : To confirm the presence of the ligated product containing all three caffeine biosynthesis genes
Results : We ran the ligation of the three genes (done July 24) on a 1% agarose gel. Three faint bands were seen at ~1.2kb, ~2.5kb, and ~3.2kb. The band at 3.2 kb indicates our desired product is present. However, gel extraction will be needed to isolate 3.2-kb product from the others.
Amplifying the Stitched Product using PCR
Experimentor : Grace Yi, Liz Geum
Aim : To amplify the three-gene product using PCR
Results : The same PCR conditions were used as for PCR 2 but with a longer extension time. The forward primer for part H and reverse primer for part L were used. Gel picture showed a very strong band at 1.2 kb region. We suspect that this is because the three caffeine genes are similar at the N and C termini likely due to sequence conservation and strep tags on the 3' end. This would enable the primers to amplify each gene individually, two adjacent genes, or all three genes (the last of which is desired). It would be thereby giving amplification of 1kb. To reach a high enough concentration for cloning, we decided to set up a larger volume reaction for the digest and ligation, gel extract at 3kb band, and clone.
Analyzing Sequencing Results
Experimentor : Grace Yi, Liz Geum
Aim : To analyze the sequencing results for PCR 3 products cloned into pSB1C3
Results : Sequencing results for PCR 3 product of each gene in pSB1C3 arrived today. With our new insert in the vector, sequencing results should show that XbaI and SpeI sites are regenerated. However, none had XbaI or SpeI cutsites, and the two other standard cutsites were present. This indicated formation of X-S scars. With clustal, we found that our sequences and those of the Munich parts do not align. We concluded that all plasmids sent in for sequencing self-ligated with high efficiency.
July 26
Creating More PCR 2 Stock
Experimenter: Grace Yi, Liz Geum
Aim : To create more PCR 2 stock for downstream reactions
Results : Optimized conditions for PCR 2 reactions from July 17 were replicated. Products were run on an agarose gel and confirmed to be the desired products based on size.
July 29
Stitching PCR 2 products for cassette assembly
Experimentor : Liz Geum
Aim : To digest the PCR 2 products with stitching enzymes and ligate for cassette assembly
Results : PCR 2 products were cut with the following the following enzymes :
H : XbaI and NcoI
J : NcoI and BamHI
L : BamHI and SpeI
To increase the efficiency of ligation reaction, 240ng of each gene was used per 20ul ligation, and ligation was left at room temperature overnight.
July 30
Gel Extraction of 3kb band
Experimentor : Liz Geum
Aim : To run and extract at 3kb band in order to acquire a purified insert and improve efficiency of ligation into X/S-cut vector.
Results : There were three bands present: brightest at 1.1kb and faint bands at 2kb and 3kb. 3kb band was gel extracted and spun down at maximum rpm for 10 minutes in purification columns acquired from Ray. The insert was quantified at 5.4ng/ul.
July 31
Ligation of 3kb stitched insert into pSB1C3
Experimentor : Liz Geum
Aim : To ligate the 3kb insert into empty pSB1C3.
Results : Ligation was set up at 1:6 vector to insert ratio. The 3kb insert was ligated with X/S cut empty vector. The ligation was left at room temperature overnight for maximum efficiency.
August 1
Transformation of yesterday's ligation product
Experimentor : Liz Geum
Aim : To transform the ligation product (3kb stitched part in vector)
Results : Chemically competent cells were transformed. We will confirm the results tomorrow with colony PCR.
August 2
Colony PCR
Experimentor : Liz Geum
Aim : To amplify the region between VF2 and VR to confirm the presence of 3kb insert in the vector.
Results : 20 white colonies were picked after GFP screening. All colonies showed small bands of ~200 base pairs, and no 3kb band was present. We decided to further test these colonies by restriction digest analysis; additional 10 white colonies from the plate were inoculated in LB/Chlor overnight cultures.
August 5
Miniprep for restriction digest analysis
Experimentor : Liz Geum
Aim : To miniprep the overnight cultures for restriction digest analysis to look for 3kb insert.
Results : 10 cultures from yesterday were miniprepped using qiagen miniprep kit.
August 7
Restriction digest analysis of miniprep plasmids
Experimentor : Liz Geum
Aim : To digest the miniprep plasmids with XbaI and PstI to check for presence of 3kb insert as well as directionality. If band is present at 3kb, it indicates that our insert is present in the right direction. If the insert went in the wrong direction, then it will only show a 5kb linearized band as only PstI cutsite will be cut.
Results : All 10 plasmids showed a linearized band at 2kb region. This likely indicates that all plasmids are empty self-ligated vectors.
August 11
Stitching PCR 2 products for cassette assembly #2
Experimentor : Grace Yi, Liz Geum
Aim : To digest the PCR 2 products with stitching enzymes and ligate for cassette assembly. This is our second attempt.
Results : PCR 2 products were cut with the following the following enzymes :
H : XbaI and NcoI
J : NcoI and BamHI
L : BamHI and SpeI
To increase the efficiency of ligation reaction, ~300ng of each gene was used per 20ul ligation, and ligation was left at room temperature overnight.
August 12
Gel Extraction of 3kb band #2
Experimentor : Liz Geum
Aim : To run and extract at 3kb band in order to acquire a purified insert and improve efficiency of ligation into X/S-cut vector. This is our second attempt.
Results : There were three bands present: brightest at 1.1kb and faint bands at 2kb and 3kb. 3kb band was gel extracted and spun down at maximum rpm for 10 minutes in purification columns acquired from Ray. The insert was quantified at 7.0ng/ul.
Ligation of the Three-Gene Product into a pSB1C3 #2
Experimenter: Grace Yi, Liz Geum
Aim : To ligate all yesterday's three-gene ligations into freshly digested pSB1C3
Results : There was concern in the lab that the vector we had been using was contaminated. We used pSB1C3 (again with GFP between the X and S cut sites) that was freshly cut with XbaI and SpeI. 1:3 vector to insert ratio was used. No insert control was also set up to determine the background.
Transformation of 3kb stitched part #2
Experimenter: Grace Yi, Liz Geum
Aim : To transform the above ligation into chemically competent cells
Results : The ligation was transformed and plated. Tomorrow, we will inoculate cultures with selected white colonies, as well as set up colony PCR to quickly check which colonies may have vectors containing the desired insert.
August 14
Colony PCR #2
Experimentor : Liz Geum
Aim : To amplify the region between VF2 and VR to confirm the presence of 3kb insert in the vector.
Results : 20 white colonies were picked after GFP screening. All colonies showed small bands of ~200 base pairs, and no 3kb band was present. Also, the no-insert control plate showed just as many white colonies as the other plate. We concluded that there was a very high self-ligation at X and S cutsites in the vector, which outruns the 3kb stitched part to be inserted into the vector. Therefore, we decided to design new primers that will add the additional PstI cutsite at 3' end of our PCR 2 products.
Designing new primers for PCR 3
Experimentor : Ray Socha, Liz Geum, Grace Yi
Aim : To design new primers that will add additional PstI cutsite at 3' end of PCR 2 products to avoid working with X/S cutsites.
Results :
Restriction Digest with PCR 1 products
Experimenter: Grace Yi, Liz Geum
Aim : To digest the miniprepped plasmids with XbaI and SpeI
Results : If the vector contains the insert, we expect two bands - a band at around 1kb (gene size) and a band at 2kb (empty vector). If the vector has our gene but in the wrong direction (thereby creating scars), or if the vector has self-ligated, pattern corresponding to uncut, supercoiled plasmid is expected. The gel showed two bands, one at 1kb and the other at 2kb. Colony 3 of part J is expected to have our gene in the correct orientation. Tomorrow, we will send this sample for sequencing to confirm.
Colony PCR
Experimenter: Grace Yi, Liz Geum
Aim : To pick colonies for confirming the present of a single-gene insert.
Results : From yesterday's transformations, we picked three white colonies from each plate and set up a colony PCR with VF2 and VR to confirm the presence of insert. This was done in parallel with the restriction digest above. Only one of the colonies for plate J (colony 3; called J3) showed amplification at 1kb. Colony J3 was picked and 5mL culture was inoculated.
August 15
Miniprep
Experimenter: Grace Yi, Liz Geum
Aim : To miniprep each of the cultures inoculated yesterday.
Results : Overnight culture (J3) from yesterday was miniprepped.
Restriction Digest with XbaI and PstI
Experimenter: Grace Yi, Liz Geum
Aim : To digest??????????
Results : Digest with XbaI and PstI was set up to check the directionality of the insert. If the insert is in the correct orientation, we expect the digest product to show a 1kb band (gene) and a 2kb band (empty vector). If the insert had gone in the wrong orientation, it would have left a scar and would therefore only cut at P site, giving a band at 3kb (linearized plasmid).
August 16
Restriction digest of PCR 1 products with XbaI and PstI
Experimentor : Grace Yi, Liz Geum
Aim : To restriction digest H, J and L PCR 1 products with XbaI and PstI for cloning into vectors with promoters and ribosome binding site.
Results : We decided to shift direction away from “stitching” assembly because of several challenges we encountered while working with X and S cutsites. Caffeine biosynthesis cassette will be assembled via standard assembly.
PCR 1 digests were purified and quantified at the following concentration :
H : 8.9ng/µl
J : 7.5 ng/µl
L : 9.4 ng/µl
Ligation of PCR 1 product into constitutive and arabinose-inducible promoters with rbs
Experimentor : Grace Yi, Liz Geum
Aim : To ligate H, J and L PCR 1 products with constitutive (const.) and arabinose-inducible promoters (pBad) as well as ribosome binding site for bacterial expression.
Results : Ligation was set up at 1:3 insert to vector ratio. Ligations reaction was left at room temperature for 2 hours, then heat inactivated.
Transformation of PCR 1 ligation
Experimentor : Grace Yi, Liz Geum
Aim : To transform PCR 1 ligation product.
Results : Competent cells were transformed We will confirm the success of transformation with cPCR next day.
August 17
Colony PCR on pBad+rbs+PCR 1 and const.+rbs+PCR 1 transformation
Experimentor : Grace Yi, Liz Geum
Aim : Colony PCR with VF2 and VR to confirm successful transformation with pBad+rbs+PCR 1 and const+rbs+PCR 1 ligation products for all three genes. Also, the amplicons need to be restriction digested and terminator has to be added to start standard assembly.
Results : Gel confirmation showed colonies with desired bands (1.2kb) for all three genes. Successful colonies were inoculated and cultured overnight for miniprep. The amplicons were purified and will be set up for restriction digest next day.
August 18
Miniprep of promoter+rbs+PCR 1
Experimentor : Grace Yi, Liz Geum
Aim : To isolate the factorial biobrick parts for caffeine biosynthesis cassette assembly.
Results : Qiagen Spin Miniprep kit used to miniprep overnight cultures in LB and chloramphenicol. Miniprep concentrations are as follows :
H pBad : 354.3 ng/µl
J pBad : 211.8 ng/µl
L pBad : 240,3 ng/µl
H const: 189.4 ng/µl
J const: 310.2 ng/µl
L const : 225.3 ng/µl
These plasmids will be submitted as factorial biobricks for caffeine synthesis cassette (BBa_K1129013, BBa_K1129014, BBa_K1129015, BBa_K1129016, BBa_K1129017, BBa_K1129018)
Start of Standard Assembly : Restriction Digest of colony PCR amplicon
Experimentor : Grace Yi, Liz Geum
Aim : To digest the colony PCR products from August 17 with EcoRI and SpeI for cloning into terminator.
Results : Digest products were purified and quantified at the following concentration.
H pBad : 66.5ng/µl
J pBad : 73.3ng/µl
L pBad : 56.0 ng/µl
H const: 80.6ng/µl
J const : 75.3 ng/µl
L const : 34.9 ng/µl
Standard Assembly : Ligation of promoter+rbs+PCR 1 into terminator
Experimentor : Grace Yi, Liz Geum
Aim : To ligate individual gene parts under const and pBad promoters with terminator for expression.
Results : Ligation was set up at 1:3 insert to vector ratio. Ligations reaction was left at room temperature for 2 hours, then heat inactivated.
Standard Assembly : Transformation of terminator ligation product
Experimentor : Grace Yi, Liz Geum
Aim : To transform the 6 ligation products for standard assembly.
Results : Competent cells were transformed We will confirm the success of transformation with cPCR next day.
August 19
Standard Assembly : Colony PCR on terminator ligation product
Experimentor : Grace Yi, Liz Geum
Aim : Colony PCR with VF2 and VR to confirm successful transformation of six promoter+PCR1+term ligation products.
Results : Gel confirmation showed colonies with desired bands (~1.5kb) for all six parts. Successful colonies were inoculated and cultured overnight in chloramphenicol for miniprep.
August 20
Standard Assembly : Miniprep of PCR 1 with promoter and terminator
Experimentor : Grace Yi, Liz Geum
Aim : To isolate the promoter+PCR1+terminator plasmids for standard assembly.
Results : Qiagen Spin Miniprep kit used to miniprep overnight cultures in LB and chloramphenicol. Miniprep concentrations are as follows :
H pBad, term: 267.1 ng/µl
J pBad, term: : 184.7 ng/µl
L pBad, term: : 421.1ng/µl
H const, term:: 221.8 ng/µl
J const, term:: 371.9ng/µl
L const, term: : 324.4 ng/µl
Standard Assembly : Restriction digest of miniprep products for standard assembly
Experimentor : Grace Yi, Liz Geum
Aim : To restriction digest L parts with EcoRI and XbaI, and J parts with EcoRI and SpeI for standard assembly.
Results : Digest products were purified and quantified at the following concentration.
J pBad , term: 87.2ng/µl
L pBad, term : 34.5 ng/µl
J const, term : 77.5ng/µl
L const, term : 69.1ng/µl
Standard Assembly : Ligation of J and L for standard assembly
Experimentor : Grace Yi, Liz Geum
Aim : To ligate J and L digest products for sequential assembly of caffeine-biosynthesis cassette.
Results : J pBad and L pBad were ligated, and J const and L const were ligated. Ligation was set up at 1:3 L to J ratio. Ligations reaction was left at room temperature for 2 hours, then heat inactivated.
Standard Assembly : Transformation of ligation product
Experimentor : Grace Yi, Liz Geum
Aim : To transform the two J+L ligation products for standard assembly.
Results : Competent cells were transformed We will confirm the success of transformation with cPCR next day.
August 21
Standard Assembly : Colony PCR
Experimentor : Grace Yi, Liz Geum
Aim : Colony PCR with VF2 and VR to confirm successful transformation with J+L ligation products for both pBad and constitutive promoters.
Results : Gel confirmation showed colonies with desired bands (3.5kb) for the two assembled parts. Successful colonies were inoculated and cultured overnight in LB and chloramphenicol for miniprep.
August 22
Standard Assembly : Miniprep of overnight cultures
Experimentor : Grace Yi, Liz Geum
Aim : To isolate plasmids with J+L under pBad and const promoters for standard assembly.
Results : Qiagen Spin Miniprep kit used to miniprep overnight cultures in LB and chloramphenicol. Miniprep concentrations are as follows :
J+L pBad : 211.4 ng/µl
J+L const : 248.9 ng/µl
Standard Assembly : Restriction digest of miniprep products for standard assembly
Experimentor : Grace Yi, Liz Geum
Aim : To restriction digest J+L plasmid with EcoRI and XbaI and promoter+H PCR1+term with EcoRI and SpeI in the final step of standard assembly.
Results : Digest products were purified and quantified at the following concentration :
pBad+H PCR 1+term : 59.1ng/µl
const+H PCR 1+term : 31.1ng/µl
J+L pBad : 77.5ng/µl
J+L const : 52.4 ng/µl
Standard Assembly : Ligation of H and J+L for standard assembly
Experimentor : Grace Yi, Liz Geum
Aim : To ligate H digest product with J+L in the final step of standard assembly.
Results : H pBad+term digests and H const+term digests were ligated with J+L digests under respective promoters. Ligation was set up at 1:3 J+L to H ratio. Ligations reaction was left at room temperature for 2 hours, then heat inactivated.
Standard Assembly : Transformation of assembled cassette
Experimentor : Ray Socha
Aim : To transform assembled caffeine-biosynthesis cassette product under pBad and constitutive promoters into heatshock cells.
Results : Competent cells were transformed. We will confirm the success of transformation with cPCR next day.
August 23
Standard Assembly : Colony PCR on caffeine cassette transformation
Experimentor : Ray Socha
Aim : Colony PCR with VF2 and VR to confirm successful transformation with assembled caffeine synthesis cassette under pBad and constitutive promoters.
Results : Gel confirmation showed colonies with desired bands (4.5kb) for assembled complete cassette for both promoters. Successful colonies were inoculated and cultured overnight in LB and chloramphenicol for miniprep.
August 24
Standard Assembly : Miniprep of Assembled Caffeine Cassette
Experimentor : Grace Yi, Liz Geum
Aim : To isolate the assembled cassettes under pBad and const promoters for biobrick submission.
Results : Qiagen Spin Miniprep kit used to miniprep overnight cultures in LB and chloramphenicol. Miniprep concentrations are as follows :
pBad caffeine cassette : 276.0ng/µl
const caffeine cassette : 244.8 ng/µl
These cassettes will be submitted as assembled biobricks (BBa_K1129019, BBa_K1129020).
September 1
Colony PCR
Experimentor : Grace Yi, Liz Geum
Aim : To confirm the presence of a single gene behind a constitutive promoter and bacterial ribosome site. Plates of each of the three genes were tested.
Results : A gel will be run tomorrow.
September 2
Confirmation Gel
Experimentor : Grace Yi, Liz Geum
Aim : To visualize the colony PCR products run yesterday.
Results : Fourteen of the fifteen chosen colonies showed bands at the correct size - around 1.4 kb. One of each of parts H, J, and L were picked for inoculating 5-mL LB/chloramphenicol cultures.
Colony PCR
Experimentor : Grace Yi, Liz Geum
Aim : To confirm the presence of a single gene behind an arabinose-induced promoter and bacterial ribosome site. Plates of each of the three genes were tested.
Results : The gel indicated a ~1.4 kb product for one of the J colonies and two of the L colonies. These were picked for inoculating 5-mL LB/chloramphenicol cultures.
September 3
Miniprep
Experimentor : Grace Yi, Liz Geum
Aim : To miniprep the the six cultures inoculated yesterday. These include all three of the genes behind a constitutive promoter and two of the genes behind an arabinose-induced plasmid.
Results : A Qiagen Spin Miniprep Kit was used, as before. All six products were prepared and sent for sequencing.
Colony PCR
Experimentor : Grace Yi, Liz Geum
Aim : To confirm the presence of a single gene (H) behind an arabinose-induced promoter and bacterial ribosome site. The colonies chosen yesterday did not show bands of the expected size.
Results : A gel showed a single sample with an amplified band of the expected size. This was used to inoculate a 5-mL culture. After miniprepping this, we will begin standard assembly to stitch the three genes together.
September 5
Start of Standard Assembly: PCR, Restriction Digest, and Ligation
Experimentor : Grace Yi, Liz Geum
Aim : To start standard assembly, the first step being adding a terminator to each of the six single-gene constructs. PCR was set up using primers VF2 and VR.
Results : A gel showed that amplification was successful. Each amplicon was digested with EcoRI and SpeI, and the reaction mixture purified. These products were ligated overnight into a pSB1C3 vector digested with EcoRI and XbaI and containing a terminator.
September 6
Transformation of Ligation Products
Experimentor : Grace Yi, Liz Geum
Aim : To transform yesterday’s ligation products into chemically competent cells. This would allow amplification of a plasmid containing a full cassette of promoter, ribosome binding site, gene, and terminator.
Results : The transformed cells were plated and incubated at 37ºC overnight.
September 7
Colony PCR
Experimentor : Grace Yi, Liz Geum
Aim : To confirm the presence of our desired insert in the transformed cells from yesterday. Ten colonies were picked per plate.
Results : There were samples from each plate (a promoter & gene combination) that showed bands at around the correct size, 1.5 kb. The corresponding colonies were used to inoculate overnight cultures for miniprepping.
September 8
Miniprep
Experimentor : Grace Yi, Liz Geum
Aim : To miniprep the cultures inoculated yesterday. These include one of the genes behind a constitutive or arabinose-induced promoter.
Results : A Qiagen Spin Miniprep Kit was used, as before.
Restriction Digest of Miniprep Products
Experimentor : Grace Yi, Liz Geum
Aim : To digest the two plasmids containing L (the last gene of the biosynthetic pathway) with EcoRI and XbaI, and the plasmids containing H and J with EcoRI and SpeI. This would enable the stitching of the three genes using Standard Assembly.
Results :
Ligation of parts J and L
Experimentor : Grace Yi, Liz Geum
Aim : To join the last two genes of the caffeine synthetic pathway. Parts J and L under a constitutive promoter were ligated together, as well as parts J and L under an arabinose-induced promoter. This would allow constitutive or induced expression of the entire biosynthetic pathway.
Results : The reaction was left overnight for greater efficiency.
September 17
Preparing BioBricks for shipping
Experimentor : Grace Yi, Liz Geum
Aim : To prepare biobricks (factorial and assembled) for shipping.
Results : Six individual biobricks and two assembled cassettes were prepared for shipping. Biobricks were diluted to 25ng/ µl and placed in strip-tubes.
