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	==July 26==		==July 26==
-	===Creating more PCR 2 stock for ligation	+	===Creating more PCR 2 stock===
		+	'''Experimenter: ''' Grace Yi, Liz Geum
		+	'''Aim : '''
	===		===

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Revision as of 21:55, 31 August 2013

iGEM Home

Experimenters: Grace Yi and Liz Geum

Contents 1 Background Information: Overview of Main Reactions 2 June 20th 2.1 Making Heat-Shock Competent Cells 3 June 22th 3.1 Ordering Primers for PCR 1 3.2 June 20 3.3 June 27 3.4 July 4 3.5 July 5 3.6 July 8 3.7 July 9 3.8 July 11 3.9 July 12 3.10 July 15 4 July 17 4.1 Optimizing PCR 2 5 July 18 5.1 Restriction digest of PCR 2 products 6 July 19 6.1 Ligation and transformation of PCR 2 7 July 22 7.1 Overnight cultures 8 July 23 8.1 Miniprep of PCR 2 transformation 9 July 24 9.1 Stitching PCR 2 products for cassette assembly 10 July 25 10.1 Checking the 3kb ligated product 10.2 Amplifying the stitched gene 11 July 26 11.1 Creating more PCR 2 stock 12 = 12.1 August 12 12.2 August 13 12.3 August 14 12.4 August 15

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
"PCR 4": to change the 5' end of the CaXMT (1st in the series) and 3' end of the CaDXMT (last in the series) to more unique sequences
"PCR 5": to amplify the three-gene ligation by binding specifically to the new 5' and 3' ends made by PCR 4

June 20th

Making Heat-Shock Competent Cells

Experimenter: Joe, Dan, Grace

Aim: To make heat-shock competent cells for upcoming transformations

Results: The 1.5mL microcentrifuge tubes were labelled "HS" on the lid and stored in the -80º Freezer #3.

June 22th

Ordering Primers for PCR 1

Experimenter: Grace and Liz

Aim: To make heat-shock competent cells for upcoming transformations

Results: The 1.5mL microcentrifuge tubes were labelled "HS" on the lid and stored in the -80º Freezer #3.

June 20

Heat-shock competent cells were made today - they are labelled "HS" on the lid and can be found in the top compartment of the -80º Freezer #3 in the freezer room. We will be ordering primers to modify the TU Munich caffeine biosynthesis BioBricks we got in the Distribution Kit. These are found in wells 17H, 17J, and 17L on plate 2.

June 27

Parts 17H/J/L were previously transformed into the above-mentioned competent cells and plated. Today, transformation efficiency was found to be very low - two colonies per plate after more than 18 hours at 37ºC. The three parts were miniprepped, with DNA concentration of 154.3ng/ul, 323.8ng/ul and 117.3ng/ul respectively.

July 4

We ran a confirmation PCR on the miniprepped plasmids with standard Biobrick primers, VF2 and VR, to confirm the presence of an insert in the backbone. 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

We acquired aliquots of "good" competent cells from Ray, our grad advisor, and re-tried the transformation of the distribution kit parts.

In order to confirm our parts of interest (17H/J/L) contained the desired insert, we retried PCR, this time with 1 uL of eluted DNA from the distribution kit as template. Even with this low concentration of template, amplification appeared successful. A bright band was seen for 17J and no bands for the other two, validating yesterday's PCR results. As our project depended largely on the manipulation of the inserts in each part, we sent them for sequencing to confirm what PCR told us. The results revealed the correct insert was indeed present in each of the three parts, a "green light" to moving forward.

We designed and ordered primers to remove the "TCACA" yeast consensus sequence found in the TU Munich Biobrick parts, as 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 8

Transformation of 17H/J/L with Ray's competent cells was found to be highly efficient, indicating our own heat-shock competent cells were faulty. We received the protocol used to prepare these cells, and a new stock was made today.

July 9

Cultures of 17H/J/L were miniprepped, and with the products, we:

• 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)

The confirmation gel for the restriction digest showed there was an insert slightly larger than 1kb for each of the genes, which was desired. However, 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 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.

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 in preparation for ligation.

July 12

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 to avoid illegal cut sites. 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 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. The product was cut with XbaI and SpeI.

PCR 1 product for parts J and L were cut again with XbaI and SpeI with an increased amount of DNA, as a gel 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

Checking the 3kb ligated product

Experimentor :" Grace Yi, Liz Geum
"Aim : " To confirm the presence of the ~3kb ligated product
"Results : " 3 faint bands at ~1.2kb, ~2.5kb, and ~3.2kb were visible.

Amplifying the stitched gene

Experimentor :" Grace Yi, Liz Geum
Aim : To amplify the 3kb stitched gene for cloning
Results : Same PCR conditions were used as for PCR 2 with longer extension time with Forward primer for gene 1 and reverse primer for gene 3. 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 N and C terminus likely due to the strep tags, and the primers can act as a full primer set for all three genes, 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.

July 26

Creating more PCR 2 stock

Experimenter: Grace Yi, Liz Geum Aim :

=

August 12

We transformed three vectors into heat-shock cells: each gene (H/J/L) individually in pSB1C3.

We also set up another massive ligation with the remainder of our X/S-cut PCR 2 products. The goal was to run an gel and gel extract the band at 3kb, which would contain the three-gene ligation we are looking for. The band at 3kb was very faint on the gel, and after extraction the concentration of DNA in the eluted sample was found to be around 10 ng/uL.

August 13

White colonies from yesterday's transformations (with the vector thought to be faulty) were picked and 5mL cultures inoculated for miniprepping tomorrow.

Yesterday's ligations (yielding a 3-gene product), as they had previously been digested with XbaI and SpeI, were ready to ligated into a vector. These were ligated into pSB1C3 (again with GFP between the X and S cut sites) that was freshly cut with X and S in case the vector we had previously been using was faulty. Eight new vectors were created: parts H, J, L individually in pSB1C3 (3 vectors), yesterday's 3-gene product in pSB1C3 with 3:1 and 6:1 insert to vector ratios (2 vectors), and remaining 3-gene product from several days ago also with the different insert:vector ratios (2 vectors). We set up ligation with X/S-cut PCR 2 products into pSB1C3 to create individual biobricks. There was also a "no insert control" to determine how many colonies form from the spontaneous closing of the pSB1C3 cut with X and S. These were all 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

Overnight cultures from yesterday were miniprepped (6 samples for each gene).

Then, we digested the miniprepped plasmids with XbaI and SpeI. 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, uncut plasmid pattern on gel is expected.

From yesterday's transformations, we picked 3 white colonies from each plate and set up a colony PCR with VF2 and VR to confirm the presence of insert. Only one of the colonies for plate J (J3) showed amplification at 1kb. Colony J3 was picked and 5mL culture was inoculated.

August 15

Overnight culture (J3) from yesterday was miniprepped and set up for digest with XbaI and PstI 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). Gel showed two bands, one at 1kb and the other at 2kb. J3 is expected to have our gene in the correct orientation. Tomorrow, we will send this sample for sequencing to confirm.