Question

I don't know how to distinguish between the coding/noncoding strand on the problem. I have to put fragment into cut segment and then determine what happened when that occurred and why the experimenter is receiving the observations she's getting.

Please do ligation, transcription and translation.

(IV). A cloning vector is cut with the restriction endonuclease Sma 1, whose restriction site is

C C C (cut in between here, as shown in image) G G G

and treated with alkaline phosphatase to prevent the relegation of the plasmid. The synthetic,

double-stranded DNA fragment (foreign DNA) with sequence

5' -G G A C T T A C T A C C C A A G T A- OH 3'

3' OH-C C T G A A T G A T G G G T T C A T- 5'

is inserted into the Smal site of the vector, whose location with respect to the beta-galactosidase gene in the cloning vector is shown below. (it is a blunt-end ligation!)

+H3N-Met - Thr - Met - Ile - Thr - Asn - Ser - Arg - Gly ---> (functional beta-galactosidase gene Starts here.)

- ATG | ACC | ATG | ATT | ACG | AAT | TCC | C(arrow points here to cut here) GG | GGA -

↑

Cut here

Upon transformation of the ligation mixture into E. coli and in the presence of the indicator X-gal, 52% of the plaques on the plate are blue and 48% are clear. Give the most likely explanation for this result. (Blue colonies are not due to relegation of the plasmid!!)

SHOW ALL YOUR WORK! (Include ligation, transcription and translation of the ligated products).

HINT: Make sure to identify the coding/non-coding strands!!! You should refer to lab 7 power points for this!

You may need more room to work on this! Please attach your answer or write on the back of this page. PLEASE be NEAT!!!!

(IV). A cloning vector is cut with the restriction endonuclease Sma 1, whose restriction site is ССС GGG and treated with alkaline phosphatase to prevent the relegation of the plasmid. The synthetic, double-stranded DNA fragment (foreign DNA) with sequence ·G G A C T T A C T A C C C A A G T A-OH 3. OH-CCTGAATGATGGGTTCAT 5' is inserted into the Smal site of the vector, whose location with respect to the β-galactosidase gene in the cloning vector is shown below. it is a blumt-end ligation! H,N-Met-Thr-Met . Ile -Thr . Asn . Ser . Arg -Gly→ finctionalp- galactosidase gene Starts here. Upon transformation of the ligation mixture into E. coli and in the presence of the indicator X-gal, 52% of the plaques on the plate are blue and 48% are clear. Give the most likely explanation for this result. (6 points) SHOW A for this You may need more room to work on this! Please attach your answer or write on the back of this page, PLEASE be NEAT (IV). Acloning vector is cut with the restriction endonuclease Sma, whose restriction site is and treated with alkaline phosphatase to prevent the relegation of the plasmid. The synthetic double-stranded DNA fragment (foreign DNA) with sequence 5 -GGACTTACTACCCAAGT A- OH 3' 3 OH-C CTGAATGATGGGTTCAT- 5" is inserted into the Smal site of the vector, whose location with respect to the β-galactosidase gene in the cloning vector is shown below. i is a blunt-end ligation!) H,N-Met-Thr-Met - Ile -Thr - Asn - Ser -Arg - Gly→ functional β- galactosidase gene Starts here. -ATG ACC ATG ATT ACG AAT TCC cGG GGA- ut her Upon transformation of the ligation mixture into E. coli and in the presence of the indicator X-gal, 52% of the plaques on the plate are blue and 48% are clear. Give the most likely explanation for this result. (6 points) (Blue colonies are not due to relegation of the plasmid!! SHOW ALL YOUR WORK! Include ligation, transcription and translation of the ligated products HINT: Make sure to identify the coding/non-coding strands!!! You should refer to lab 7 power points You may need more room to work on this! Please attach your answer or write on the back of this page . PLEASE be NEAT/!!! Third Position 3-end Coding rad Template and Secand Position DNA UUU Phe UCU Ser UAUTyyUGU C UUC Phe UCC Ser UACT UGC Cs UUA LeuUCA Ser UAA Stop UGA Stop RNA polymerase CUU LeuCCU Pro CAU His CGUArg CUC Leu COC Po CACHs CGC Arg CUA Leu CCA Pro CA GIn CGAArg RNa transcript UU AU AUA Ile MAThr AAA Lys AGA Arg AUG Met ACGThr AGLs AGG Arg GUU VlGCU Ala GAU Asp GGU Gly GUC Val GCC Ala GMAp GGC Gly GUA ValGGAAla GAA GhGGA Gly

Answer 1

The blue-white screening

The transformed bacteria can be screened by choosing a vector and E. coli strain that is compatible with blue/white screening, which takes advantage of intracistronic α-complementation to regenerate β-galactosidase activity. Many E. coli strains used for cloning and propagation of plasmids contain a chromosomal deletion of the lac operon but carry an F´ episome that provides the remaining coding sequence of the lacZ gene. The functional lacZ gene product, β-galactosidase, is produced when the lacZ coding information missing on the F´ episome is provided by the information contained in the plasmid. This activity is detected by plating bacteria transformed by plasmids on plates containing isopropyl β-D-thiogalactopyranoside (IPTG; an inducer of the lac promoter) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal; a dye that produces a blue colour when hydrolyzed by β-galactosidase). When the reading frame of the α- peptide is disrupted by insertion of a foreign DNA fragment or deletion of vector sequences, α-complementation does not occur, and the bacterial colonies remain white or occasionally light blue.

But here blue colonies are not due to relegation of the plasmid.

The answer to this question is describes in a stepwise manner.

1. The enzyme SmaI cuts the DNA at CCCGGG site and produces blunt ends.
2. So, the insert can be ligated with the vector in the following two ways:

5' -G G A C T T A C T A C C C A A G T A- OH 3'

3' OH-C C T G A A T G A T G G G T T C A T- 5'

or

5' -TACTTGGGTAGTAAGTCC- OH 3'

3' OH-ATGAACCCATCATTCAGG- 5'

3. The vector sequence is :

+H3N-Met - Thr - Met - Ile - Thr - Asn - Ser - Arg - Gly ---> (functional beta-galactosidase gene Starts here.)

- ATG | ACC | ATG | ATT | ACG | AAT | TCC | CGG | GGA -

4. Now, look into the 1^st option in point 2. If the DNA is inserted that way, the vector insert sequence would look like:

5’ ATG | ACC | ATG | ATT | ACG | AAT | TCC | CGG | ACT | TAC | TAC | CCA | AGT| AGG| GGA – OH 3’

The insert sequence is written in bold.

The corresponding mRNA sequence (Transcription) is

5’ AUG | ACC | AUG | AUU | ACG | AAU | UCC | CGG | ACU | UAC | UAC | CCA | AGU| AGG| GGA – OH 3’

The corresponding amino acid sequence is:

+H3N-Met - Thr - Met - Ile - Thr - Asn - Ser - Arg – Thr – Tyr – Tyr – Pro – Ser – Arg - Gly ---> (functional beta-galactosidase gene Starts here.)

The insert does not change the coding frame for the beta-galactosidase, so the colony having the insert in this orientation will have a functional beta-galactosidase enzyme. Thus, the colonies will form blue colouration.

5. Now, look into the 2^nd option in point 2. If the DNA is inserted that way, the vector insert sequence would look like:

5’ ATG | ACC | ATG | ATT | ACG | AAT | TCC | CTA | CTT | GGG | TAG | TAA | GTC| CGG| GGA – OH 3’

The insert sequence is written in bold.

The corresponding mRNA sequence (Transcription) is

5’ AUG | ACC | AUG | AUU | ACG | AAU | UCC | CUA | CUU | GGG | UAG | UAA | GUC| CGG| GGA – OH 3’

The corresponding amino acid sequence is:

+H3N-Met - Thr - Met - Ile - Thr - Asn - Ser - Arg – Leu – Leu – Gly – stop ---> (non functional beta-galactosidase gene.)

The insert changes the coding frame for the beta-galactosidase by introducing a stop codon in the coding frame. The peptide generates with this insert is shorter in size. So the colony having the insert in this orientation will have non-functional beta-galactosidase enzyme. Thus, the colonies will form white/ transparent colouration.

6. As per the question, the blue coloured colony is 52% and the white colony is of 48%. The chances of each of these two ligation events are 50%. So, almost 50% of the colony will show blue colour and 50% is clear.

Kindly revert for any queries and concerns.